Neutrophil infiltration regulates clock-gene expression to organize daily hepatic metabolism

Liver metabolism follows diurnal fluctuations through the modulation of molecular clock genes. Disruption of this molecular clock can result in metabolic disease but its potential regulation by immune cells remains unexplored. Here, we demonstrated that in steady state, neutrophils infiltrated the mouse liver following a circadian pattern and regulated hepatocyte clock-genes by neutrophil elastase (NE) secretion. NE signals through c-Jun NH2-terminal kinase (JNK) inhibiting fibroblast growth factor 21 (FGF21) and activating Bmal1 expression in the hepatocyte. Interestingly, mice with neutropenia, defective neutrophil infiltration or lacking elastase were protected against steatosis correlating with lower JNK activation, reduced Bmal1 and increased FGF21 expression, together with decreased lipogenesis in the liver. Lastly, using a cohort of human samples we found a direct correlation between JNK activation, NE levels and Bmal1 expression in the liver. This study demonstrates that neutrophils contribute to the maintenance of daily hepatic homeostasis through the regulation of the NE/JNK/Bmal1 axis.


Introduction
Circadian rhythms regulate several biological processes through internal molecular mechanisms (Dibner et al., 2010) and the chronic perturbation of circadian rhythms is associated with the appearance of metabolic syndrome (Kolla and Auger, 2011). This homeostasis is closely dependent on the circadian system in the liver, which shows rhythmic expression of enzymes associated with glucose and lipid metabolism (Haus and Halberg, 1966;North et al., 1981;Tahara and Shibata, 2016). Moreover, mice with mutations in clock genes encoding nuclear receptors have impaired glucose and lipid metabolism and are susceptible to diet-induced obesity and metabolic dysfunction, consistent with the idea that these genes control hepatic metabolic homeostasis (Delezie et al., 2012;Kudo et al., 2008;Lamia et al., 2008;Rey et al., 2011;Tong and Yin, 2013;Turek et al., 2005;Yang et al., 2006). Besides, recent reports have shown that hepatic physiology follows a diurnal rhythm driven by clock genes, with expression of proteins involved in fatty acid synthesis higher in the morning while those controlling fatty acid oxidation are higher at sunset (Toledo et al., 2018;Zhou et al., 2015).
Blood leukocyte levels also oscillate diurnally, as does the release of hematopoietic stem cells and progenitor cells from the bone marrow (BM) (Haus and Smolensky, 1999;Lucas et al., 2008;Méndez-Ferrer et al., 2008) and their recruitment into tissues (Adrover et al., 2019;He et al., 2018;Scheiermann et al., 2012). Oscillatory expression of clock genes in peripheral tissues is largely tuned by the suprachiasmatic nucleus (Dibner et al., 2010;Druzd and Scheiermann, 2013;Huang et al., 2011;Reppert and Weaver, 2002); however, the potential regulation of daily rhythms of specific tissues by immune cells remains largely unexplored, both in steady state and during inflammation. Although the molecular mechanisms linking circadian rhythms and metabolic disease are largely unknown, several studies have demonstrated a strong association between leukocyte activation and metabolic diseases (McNelis and Olefsky, 2014). A prime example is the BM, where engulfment of infiltrating neutrophils by tissue-resident macrophages modulates the hematopoietic niche (Casanova-Acebes et al., 2013).
Here, we demonstrate that circadian neutrophil infiltration into the liver controls the expression of eLife digest Every day, the body's biological processes work to an internal clock known as the circadian rhythm. This rhythm is controlled by 'clock genes' that are switched on or off by daily physical and environmental cues, such as changes in light levels. These daily rhythms are very finely tuned, and disturbances can lead to serious health problems, such as diabetes or high blood pressure.
The ability of the body to cycle through the circadian rhythm each day is heavily influenced by the clock of one key organ: the liver. This organ plays a critical role in converting food and drink into energy. There is evidence that neutrophils -white blood cells that protect the body by being the first response to inflammation -can influence how the liver performs its role in obese people, by for example, releasing a protein called elastase. Additionally, the levels of neutrophils circulating in the blood change following a daily pattern. Crespo, Gonzá lez-Terá n et al. wondered whether neutrophils enter the liver at specific times of the day to control liver's daily rhythm.
Crespo, Gonzá lez-Terá n et al. revealed that neutrophils visit the liver in a pattern that peaks when it gets light and dips when it gets dark by counting the number of neutrophils in the livers of mice at different times of the day. During these visits, neutrophils secreted elastase, which activated a protein called JNK in the cells of the mice's liver. This subsequently blocked the activity of another protein, FGF21, which led to the activation of the genes that allow cells to make fat molecules for storage. JNK activation also switched on the clock gene, Bmal1, ultimately causing fat to build up in the mice's liver. Crespo, Gonzá lez-Terá n et al. also found that, in samples from human livers, the levels of elastase, the activity of JNK, and whether the Bmal1 gene was switched on were tightly linked. This suggests that neutrophils may be controlling the liver's rhythm in humans the same way they do in mice.
Overall, this research shows that neutrophils can control and reset the liver's daily rhythm using a precisely co-ordinated series of molecular changes. These insights into the liver's molecular clock suggest that elastase, JNK and BmaI1 may represent new therapeutic targets for drugs or smart medicines to treat metabolic diseases such as diabetes or high blood pressure. clock genes through the regulation of c-Jun NH2-terminal kinase (JNK) and the hepatokine fibroblast growth factor 21 (FGF21), driving adaptation to daily metabolic rhythm.

Rhythmic neutrophil infiltration into the liver modulates the expression of hepatic clock genes
Virtually all cell types have an internal clock that controls their rhythmicity through the periodic expression of clock genes (Robles et al., 2014;Tahara and Shibata, 2016). However, it is unknown how these multiple cell rhythms are integrated. The liver is an essential metabolic organ that controls body glucose and lipid homeostasis (Manieri and Sabio, 2015), and neutrophil infiltration alters its function (González-Terán et al., 2016). We hypothesized that the metabolic cycles in the liver might be entrained by rhythmic neutrophil infiltration. To test this, we harvested liver, BM, and blood from C57BL6J mice at 4 hr intervals over a 24 hr period. Liver neutrophil infiltration showed a clear diurnal pattern, with a peak at ZT2, coinciding with liver-driven lipogenesis in mice (Zhou et al., 2015), and a nadir during the night, at ZT14 ( Figure 1A), correlating with lipolysis (Zhou et al., 2015). These oscillations corresponded directly to changes in neutrophil numbers in blood (Figure 1-figure supplement 1A), suggesting that liver infiltration might result from higher neutrophil migration to the liver. We first confirmed that neutrophils were infiltrated in the liver using 3D microscopy. According to published data (Casanova-Acebes et al., 2018), infiltrated neutrophils presented an intrasinusoidal distribution in the liver, different to that observed in the Kupffer cells population ( Figure 1B and Figure 1-figure supplement 1B). Then we evaluated whether myeloid chemokines could be involved in circadian neutrophil recruitment into the liver. Analysis of liver lysates indicated that the expression of the hepatocyte-derived neutrophil chemoattractant Cxcl1 (Su et al., 2018) was higher at ZT2 than a ZT14. Moreover, mRNA of Cxcl1 in liver samples showed the same oscillation pattern than infiltrated neutrophils, suggesting that this chemokine may be important in the regulation of the neutrophil diurnal cycle (Figure 1-figure supplement 1C).
The infiltration pattern correlated with liver expression levels of the clock-gene Bmal1, peaking at ZT2 and bottoming at ZT14 ( Figure 1C). Infiltration also correlated inversely with the expression of Nr1d2 (encoding Rev-erb b), Per2, and Cry2 ( Figure 1C), which are important proteins in the control of circadian rhythms (Reppert and Weaver, 2002), consistent with the feedback loop that controls their expression. Bmal1 is thought to induce lipogenesis (Zhang et al., 2014), whereas Nr1d2 controls lipid metabolism and its reduced expression promotes lipogenesis and steatosis (Delezie et al., 2012;Solt et al., 2012). In agreement with these studies, liver triglycerides were higher at ZT2 than at ZT14 ( Figure 1D).
Our results show a correlation between neutrophil infiltration, hepatocyte Bmal1 expression, and lipid metabolism regulation, raising the possibility that neutrophils signal to hepatocytes to modulate the expression of circadian genes. Exposure of mouse hepatocytes in vitro to freshly isolated neutrophils increased hepatocyte expression of the clock genes Bmal1 and Clock. In contrast, no effect was observed upon exposure to T or B lymphocytes, or macrophages, suggesting the existence of a neutrophil-to-hepatocyte communication that controls hepatocyte clock-gene expression ( Figure 1E and Figure 1-figure supplement 1D).
We then investigated whether neutrophil elastase (NE), a proteolytic enzyme reported to regulate liver metabolism, could regulate hepatocyte clock genes (Mansuy-Aubert et al., 2013;Talukdar et al., 2012). Exposure to elastase reproduced the same increase in hepatocyte Bmal1 and Clock expression in contrast with another protease that did not affect Bmal1 expression ( Figure 1F and Next, neutrophil-mediated regulation of liver clock-gene expression in vivo was investigated using a previously characterized genetic model of neutrophil deficiency (Dzhagalov et al., 2007;Steimer et al., 2009;  Low hepatic neutrophil infiltration in neutropenic mice correlated with reduced expression of Bmal1 and Clock ( Figure 1G) and increased expression of Cry2 and Per2 at ZT2 ( Figure 1G). These changes in clock-gene expression were accompanied by lower liver triglyceride levels ( Figure 1H). Furthermore, lack of neutrophils perturbed the diurnal rhythmicity in Bmal1, Clock, and Per2 expression in the liver without affecting clock genes in other organs such as the lung, in which there is no Figure 1. Neutrophil infiltration into the liver controls hepatic clock-gene expression. (A) Flow cytometry analysis of the CD11b + Ly6G + liver myeloid subset, isolated from C57BL6J mice at the indicated ZTs. Left, CD11b + Ly6G + liver myeloid subset analyzed at 6 hr intervals and normalized by the tissue weight. Right, percentage of CD11b + Ly6G + population analyzed at 4 hr intervals and normalized to ZT2 (n = 5). (B) Representative 3-D image of liver section showing the distribution on infiltrated neutrophils. Livers were stained with anti-S100A9 (Mrp14) (red) and vessels were stained with anti- Figure 1 continued on next page correlation between the peak of neutrophil infiltration and Bmal1 expression (Figure 1-figure supplement 2D,E). Our results thus indicate that neutrophils might specifically control the expression of hepatocyte circadian clock genes in steady state.

Disruption of daily neutrophil infiltration in the liver affects hepatocyte molecular clock and metabolism
Chronic jet lag alters liver circadian genes and disrupts liver metabolism (Kettner et al., 2016). Analysis of a mouse model of jet lag revealed complete disruption of the circadian liver neutrophil infiltration with increased hepatic neutrophil infiltration even at ZT14 (Figure 2A). Abolition of rhythmic neutrophil hepatic infiltration under jet lag correlated with increased steatosis and high levels of liver triglycerides ( Figure 2B). To evaluate whether the metabolic effect of circadian perturbation was caused by the increased neutrophil infiltration, we exposed neutropenic and control mice to the jet lag protocol (Figure 2-figure supplement 1A,B). Jet lag-induced steatosis was less severe in neutropenic mice ( Figure 2C), and disruption of diurnal liver expression of Bmal1 detected in control jet-lagged mice was partially ablated in neutropenic mice ( Figure 2D). Similar results were also observed in mice with impaired neutrophil migration such as Cxcr2 MRP8-KO BM transplanted mice (Eash et al., 2010;Mei et al., 2012) and p38g/d Lyzs-KO mice (González-Terán et al., 2016). In both models, the reduction of neutrophil infiltration correlated with decreased levels of liver Bmal1 expression and protection from jet lag-induced steatosis (Figure 2-figure supplement 1C-G). These results are consistent with the role of neutrophils in the control of liver clock genes.
Inflammation plays a key role in the pathogenesis of non-alcoholic fatty liver disease (Tiniakos et al., 2010) and the development of hepatic steatosis is associated with increased liver infiltration by myeloid cells, particularly neutrophils (González-Terán et al., 2016;Mansuy-Aubert et al., 2013;Talukdar et al., 2012;Tiniakos et al., 2010). Two widely used mouse models of hepatic steatosis, high-fat diet (HFD) and methionine-choline-deficient (MCD) diet, increased liver neutrophil infiltration in WT mice at ZT2, ZT14, and ZT18 ( Figure 2E,F). Consistent with a neutrophil-to-hepatocyte communication in the regulation of hepatocyte clock genes, the MCD diet enhanced Bmal1 expression and inhibited Cry2 and Per2 expression in control mice, but not in neutropenic mice at ZT2 ( Figure 2G). Altered liver clock-gene regulation in neutropenic mice was associated with protection against steatosis and lower liver triglycerides ( Figure 2H). To confirm the role of neutrophils in modulating liver clock genes, we depleted neutrophils by injecting anti-Ly6G antibody into MCD diet-fed mice (González-Terán et al., 2016). Anti-Ly6G administration for 7 days reduced circulating neutrophil levels without affecting monocytes (  x 510 x 28 mm and 160 x 160 x 28 mm, respectively. (C) qRT-PCR analysis of circadian clock-gene and nuclear-receptor mRNA expression in livers from C57BL6J mice at the indicated ZTs (n = 5). (D) Liver triglycerides and oil-red-stained liver sections prepared from C57BL6J mice at ZT2 and ZT14. Scale bar, 50 mm (n = 5). (E) qRT-PCR analysis of clock-gene mRNA in hepatocyte cultures exposed to freshly isolated FMLP-activated neutrophils (n = 4-6 wells of 3 independent experiments). (F) qRT-PCR analysis of clock-gene mRNA in hepatocyte cultures treated with 5 nM elastase (n = 3-4 wells of 3 independent experiments). (G) qRT-PCR analysis of clock-gene and nuclear-receptor mRNA expression in livers from control mice (Mrp8-Cre) and neutropenic mice (MCL1 Mrp8-KO ) sacrificed at ZT2 (n = 5). (H) Hepatic triglycerides detected in livers from control mice (Mrp8-Cre) and neutropenic mice (MCL1 Mrp8-KO ) at ZT2 (n = 5). Data are means ± SEM from at least 2 independent experiments. *p<0.05; **p<0.01; ***p<0.005 (      and control and neutropenic (MCL1 Lyzs-KO ) mice (C-D) were housed for 3 weeks with a normal 12 hr: 12 hr light/dark cycle (Normal Cycle) or with the dark period extended by 12 hr every 5 days (JetLag). Samples were obtained at the indicated ZTs. (A) Left, flow cytometry analysis of the CD11b + Ly6G + liver myeloid subset. Data represents the percentage CD11b + Ly6G + normalized to Normal Cycle ZT2. Right, circulating neutrophils in whole blood. (n = 5-8). (B) Liver triglycerides and representative oil-red-stained liver sections at ZT14. Scale bar, 50 mm (n = 9-10). (C) Hepatic triglyceride content analyzed at 6 hr intervals, and representative oil-red-stained liver sections at ZT14. Scale bar, 50 mm (n = 4-6). (D) qRT-PCR analysis of Bmal1 mRNA in livers. (n = 5-8). (E) Flow cytometry analysis of the CD11b + Ly6G + liver myeloid subset isolated at 6 hr intervals from C57BL6J mice fed a ND, a HFD (8 weeks) or a MCD (3 weeks). The chart shows the CD11b + Ly6G + population as a percentage of the total intrahepatic CD11b + leukocyte population normalized to ND group at ZT2 (n = 5 to 10). (F-I) Control mice (Lyzs-Cre) and neutropenic mice (MCL1 Lyzs-KO ) or p38g/d Lyzs-KO were fed a ND or the MCD diet for 3 weeks and sacrificed at ZT2. (F) Representative images of the infiltration of neutrophils in the liver stained with anti-Mrp14 (blue) and anti-NE (red); nuclei with Sytox Green. Scale bar, 50 mm (Top) and 25 mm (Bottom). (G) qRT-PCR analysis of clock-gene expression in livers (n = 6). (H) Liver triglycerides and representative oil-red-stained liver sections. Scale bar, 50 mm (n = 7-6). (I) qRT-PCR analysis of clock genes in livers at ZT2 (n = 9-17). Data are means ± SEM from at least two independent experiments. *p<0.05; **p<0.01; ***p<0.005 (A to D) t-test or Welch's test. (E) Two-way ANOVA with Fisher's post hoc test; p<0.05 ND vs HFD; p<0.0001 ND vs MCD. *p<0.05; ***p<0.005 (G to I) t-test or Welch's test. ZT2 point is double plotted to facilitate viewing. The online version of this article includes the following source data and figure supplement(s) for figure 2: Source data 1. Raw data and statistical test.     To further support the role of neutrophil liver infiltration in the regulation of liver clock genes and hepatic lipogenesis during diet-induced steatosis, we leveraged a mouse model (p38g/d Lyzs-KO ) that exhibits deficient neutrophil migration and subsequently, reduced liver neutrophil infiltration after MCD diet (González-Terán et al., 2016). Compared with diet-matched control (Lyzs-Cre) mice, MCD-diet-fed p38g/d Lyzs-KO mice showed hepatic down-regulation of Bmal1, which was associated with higher expression of Cry2, and Per2 ( Figure 2I). These results suggest that the reduced neutrophil infiltration in mice lacking myeloid p38g/d expression is responsible for the altered expression of circadian clock genes. Overall, these findings strongly support that neutrophil infiltration modulates clock-gene expression in the liver, with downstream effects on liver metabolism.

Regulation of daily hepatic metabolism by neutrophils through JNK-FGF21 axis
It has been suggested that JNK activation in the liver may be regulated in a circadian manner with a peak at noon (Robles et al., 2014). To evaluate whether neutrophils might mediate this diurnal regulation of JNK, we analyzed JNK activation in neutropenic mice. Lack of neutrophils was associated with lower liver expression and activation of JNK, lower activation of the JNK downstream effector c-Jun, and lower expression of acetyl-CoA carboxylase (Acaca), a key enzyme in metabolic regulation (acetyl-CoA carboxylase; ACC) that mediates inhibition of beta-oxidation and activation of lipid biosynthesis ( Figure 3A and Figure 3-figure supplement 1A). Similar results were found in p38g/d Lyzs-KO mice, in which reduced liver neutrophil infiltration was associated with decreased JNK phosphorylation and ACC protein levels ( Figure 3B and Figure 3-figure supplement 1B). Moreover, neutrophil-treated hepatocytes showed increased JNK activation together with increased levels of ACC expression (Figure 3-figure supplement 1C). NE represents a potential mediator of this neutrophil function because elastase-treated hepatocytes also showed higher JNK activation, suggesting that this protease modulates the expression of the clock genes through the JNK signaling pathways ( Figure 3C and Figure 3-figure supplement 1D). This JNK activation was accompanied by increased Bmal1 expression ( Figure 3D), indicating that neutrophils altered liver clock-gene expression through the elastase-JNK pathway.
Our results suggest that neutrophil-mediated JNK activation might modulate hepatocyte clock genes and metabolism through the regulation of ACC. Supporting this hypothesis, specific JNK depletion in hepatocytes downregulated Bmal1, Clock, and Acaca compared to Alb-Cre ( Figure 3E and Figure 3-figure supplement 1E). According to these results, JNK inhibition reduced the expression of Bmal1, Clock and Acaca in WT liver but not in neutropenic mice (Figure 3-figure  supplement 1F,G). These data strongly suggest that JNK activation caused by neutrophil infiltration modulates clock genes and daily metabolism in hepatocytes.
JNK is an important modulator of the expression of the hepatokine circadian regulator FGF21 (Vernia et al., 2014), which controls glucose and lipid metabolism (Fisher and Maratos-Flier, 2013;Li et al., 2013;Potthoff et al., 2012). Mice lacking JNK in hepatocytes had higher FGF21 mRNA expression ( Figure 3E). In concordance with high JNK activation, FGF21 expression was reduced in neutrophil-exposed hepatocytes (Figure 3-figure supplement 1H). Moreover, neutropenic and p38g/d Lyzs-KO mice showed increased FGF21 expression ( Figure 3F and Figure 3-figure supplement 1I,J), which was consistent with the reduced hepatocyte JNK activation in these mice.
To further define the role of FGF21 in the neutrophil-mediated regulation of liver metabolism, we suppressed FGF21 expression using two independent lentiviral shRNA vectors ( Figure 3G and Figure 3-figure supplement 1K). The protection of p38g/d Lyzs-KO mice against MCD-diet-induced alterations was abrogated by shFGF21 and these mice developed steatosis with an elevated hepatic triglyceride content ( Figure 3H,I). These data further supported the idea that neutrophil infiltration controls liver metabolism through the regulation of FGF21 expression.

Neutrophil elastase deficiency affects the expression patterns of clock genes and lipid metabolism
To formally confirm the involvement of NE in circadian clock alteration, we first evaluated the diurnal oscillation of NE levels in liver from WT mice fed a normal diet (ND). According to infiltration pattern of neutrophils in the liver ( Figure 1A), we found higher NE levels at ZT2 than at ZT14. ( Figure 4A). Next, circadian clock-gene expression in NE -/mice revealed lower Bmal1 and elevated Per2 and Cry2 expression, compared to control mice ( Figure 4B), which mimicked the behavior of neutropenic mice. In addition, NE -/mice presented lower respiratory quotient during the lights-on period than WT mice, indicating that these mice have increased fat utilization as a source of energy ( Figure 4C), supporting the data that reduced liver-neutrophil infiltration results in higher lipid oxidation. Interestingly, when fed MCD or HFD diet, NE -/mice were protected against steatosis ( Figure Figure 4H). Furthermore, under HFD, NE -/mice were also refractory to these changes as these mice maintained a pattern of clock-gene expression similar to control mice in ND (Figure 4-figure  supplement 1E).
To formally test a direct contribution of NE in the regulation of hepatic clock-gene expression and liver metabolism, we infused WT or NE -/neutrophils into neutropenic mice under the jet lag protocol ( Figure 5A). The infusion of WT neutrophils was able to increase Bmal1 expression in the liver after jet lag, while neutropenic mice infused with NE -/neutrophils presented the same levels of Bmal1 than non-infused neutropenic mice ( Figure 5B). In addition, while infusion of neutropenic mice with WT neutrophils increased steatosis, neutropenic mice infused with NE -/neutrophils presented the same levels of steatosis than control neutropenic mice ( Figure 5C,D). All these data indicate that diet or jet-lag -induced hepatic infiltration of neutrophils results in dysregulation of the liver clock, and the lack of NE is enough to protect mice against these alterations.
Finally, to evaluate the translational relevance of these findings for human physiology we quantified in human livers the expression levels for the genes encoding NE, JUN (as an indicator of JNK activation) and Bmal. Our results suggest that the levels of ELANE expression directly correlate with BMAL1 and JUN mRNA in livers from a human cohort ( Figure 5E). These correlations reinforce the idea that a rhythmic neutrophil infiltration in the liver controls the expression of clock genes through the JNK pathway activation and could be a target for therapeutic intervention during non-alcoholic fatty liver disease.

Discussion
Our analysis demonstrates that neutrophils control clock genes in the liver and that reduced neutrophil infiltration protects against jet lag and diet-induced liver steatosis by altering the expression of these temporal regulators. These findings establish neutrophils as unexpected players in the regulation of daily hepatic metabolism. Our results also demonstrate that at least part of this neutrophilinduced clock modulation is mediated by elastase. These results agree with previous data showing that NE mediates the deleterious effects of neutrophils on liver metabolism and that mice lacking NE are protected against diet-induced steatosis (Mansuy-Aubert et al., 2013;Talukdar et al., 2012). The molecular mechanism underlying this regulation involves neutrophil NE that induces activation of JNK and consequently inhibits the production of the hepatokine FGF21. The JNK pathway Figure 3 continued supplement 1D (E) qRT-PCR analysis of clock genes and Fgf21 in livers from Alb-Cre, and JNK1/2 Alb-KO mice after 3 weeks of MCD diet at ZT2 (n = 9-12). (F) Immunoblot analysis of FGF21 content in liver extracts prepared from control (Lyzs-Cre) and neutropenic (MCL1 Lyzs-KO ) mice, or from Lyzs-Cre, and p38g/d Lyzs-KO mice after 3 weeks of MCD diet sacrificed at ZT2. Immunoblot quantification is shown in Figure 3-figure supplement 1I,J. (G-I) Lyzs-Cre and p38g/d Lyzs-KO mice were injected with 2 shRNA independent clones targeting FGF21. Seven days after infection, mice were placed on the MCD diet and sacrificed after 3 weeks at ZT2. (G) Immunoblot analysis of FGF21 content in liver extracts prepared from Lyzs-Cre, p38g/d Lyzs-KO , and p38g/d Lyzs-KO mice infected with FGF21 shRNA. Immunoblot quantification is shown in Figure 3   is an important modulator of liver metabolism, and lack of JNK1 and JNK2 in hepatocytes protects against steatosis (Manieri and Sabio, 2015). Here, we also demonstrate that JNK also regulates hepatocyte clock genes and, therefore, modulates diurnal adaptation of liver metabolism.
Recently published data have demonstrated that lipogenesis is increased in the light phase, in agreement with our analysis (Guan et al., 2018). We show that neutrophil infiltration causes JNK activation down-stream of elastase secretion, a time-dependent process. Indeed, phosphoproteomic analysis of the hepatic phosphorylation network identifies JNK as a key signaling enzyme with peak activation at ZT6 (Robles et al., 2017) immediately prior to the peak of lipogenic gene expression (Guan et al., 2018). Our results suggest that neutrophils induce an accumulative activation of JNK with a peak during the day that would control the lipogenic program.
Recent evidence established that the metabolic effects of JNK in the liver are mediated by FGF21 (Vernia et al., 2016;Vernia et al., 2014). Our results now show that liver FGF21 expression can be modulated through the control of JNK by neutrophils. Reduction of FGF21 by shRNA reverted the protective effect and metabolic changes induced by reduced neutrophil infiltration. In conclusion, our results show that the diurnal oscillating migratory properties of neutrophils regulate liver function in a manner that preserves daily metabolic rhythms, and that disturbance of this rhythmicity can cause disease. These results might imply a novel mechanism of action for the potential use of clockmodulating small molecules in liver health.

Study population
For the analysis of human liver mRNA levels, individuals were recruited among patients who underwent laparoscopic cholecystectomy for gallstone disease. The study was approved by the Ethics Committee of the University Hospital of Salamanca (Spain), and all subjects provided written informed consent to participate. Patients were excluded if they had a history of alcohol use disorders or excessive alcohol consumption, chronic hepatitis C or B, or body mass index !35. Baseline characteristics of these groups are listed in Figure 5-source data 1.
Mice were housed under a 12 hr light:12 hr dark cycle (Light is on at Zeitgeber Time ZT0 and off at ZT12). For jet lag experiments, the 12 hr:12 hr dark/light cycle was disrupted by extending the dark cycle 12 hr every 5 days over 3 weeks (Kettner et al., 2016). Cxcr2 MRP8-KO chimeras were generated by exposing WT recipient mice to 2 doses of ionizing radiation (625 Gy) and reconstituting them with 5 Â 10 6 donor BM (Cxcr2 MRP8-KO ) cells injected into the tail vein.
Mice were fed a methionine-choline-deficient (MCD) diet for 3 weeks or a high-fat diet (HFD) for 8 weeks (Research Diets Inc). For neutrophil depletion, mice mini-osmotic pumps (Alzet) were implanted with anti-Ly6G antibody or saline (0.4 mg/kg per day, 21 days). For JNK inhibition Figure 4 continued bar, 50 mm (n = 10). (F) Immunoblot analysis and quantifications of JNK content and activation in liver extracts prepared from WT and NE -/-. (G) Immunoblot analysis and quantification of ACC content in liver extracts from WT and NE -/mice. (H) qRT-PCR analysis of clock-genes and nuclearreceptor mRNA expression in livers from WT and NE -/mice at ZT2 and ZT14 (n = 7-8). Data are means ± SEM from at least two independent experiments. *p<0.05; **p<0.01; ***p<0.005 (A to G) t-test or Welch's test. (H) One-way ANOVA with to Tukey's post hoc test, t-test or Welch's test. The online version of this article includes the following source data and figure supplement(s) for figure 4: Source data 1. Raw data and statistical test.   6-7). Data are means ± SEM. *p<0.05; Figure 5 continued on next page experiments, mice were intraperitoneally injected with SP600125 (15 mg/kg) (Santa Cruz Biotechnology) at ZT0. For neutrophil infusion experiments, mice were intravenously injected with 3 Â 10 6 WT or NE -/purified neutrophils each 3-4 days. Neutrophils were isolated from BM using biotinylated anti-Ly6G antibody (Clone:1A8) and streptavidin-labeled magnetic microbeads (Miltenyi Biotec).
All animal procedures conformed to EU Directive 86/609/EEC and Recommendation 2007/526/ EC regarding the protection of animals used for experimental and other scientific purposes, enacted under Spanish law 1201/2005.

Cell cultures
Hepatocytes were isolated from adult females by collagenase liver perfusion and cells were filtered through a 70 mm strainer. Hepatocytes pelleted from centrifuged Percoll gradients were plated at 4 Â 10 5 cells/well on 6-well plates coated with collagen type one and incubated at 37˚C. After 24 hr, cells were treated with 0.5 mM palmitate (Sigma-Aldrich) for 6 hr and then exposed for 1 hr to freshly neutrophils (2 Â 10 6 cells/well) in the presence of 1 mM FMLP (Sigma-Aldrich). Neutrophils were isolated from BM as described above. For some experiments, neutrophils were sorted purified form the BM using an anti-Ly6G antibody (Clone: 1A8). T and B lymphocytes were sorted purified from spleens using anti-CD3 (Clone: 145-2 C11) and anti-B220 (Clone: RA3-6B2), and bone marrow macrophages (BMDM) were differentiated as previously described (González-Terán et al., 2013). All antibodies were purchased from BD Pharmingen. Alternatively, hepatocytes were exposed 2 hr to 5 nM NE (R and D Systems) or 0.5 mg/mL of collagenase A (Roche) after palmitate treatment.

Isolation of liver-infiltrating leukocytes
Mice were perfused with 20 mL of PBS and livers were collected and dissociated. Cell suspension was passed through a 70 mm strainer and centrifuged twice at 50 xg for 2 min to discard the liver parenchyma. For some experiments, livers were incubated for 15 min with 1 mg/mL Collagenase A (Roche) and 2 U/mL DNase (Sigma) at 37˚C, and lungs were incubated for 25 min with 0,25 mg/ml Liberase TL (Sigma) and 5 U/mL DNase (Sigma) at 37˚C Leukocyte fraction was collected and stained with anti-CD45 (Clone: 30-F11), from Invitrogen, anti-CD11b (Clone: M1/70), anti-Ly6G (Clone: 1A8) or anti-Ly6C/G (Clone: RB6-8C5), from BD Pharmingen, and alternatively, with anti-F4/80 (Clone: BM8), from Invitrogen, and Goat anti-Clec4F from R and D Systems and conjugated with anti-goat Alexa 647. Cells were sorted on a FACSAria to >95% purity. Flow cytometry experiments were performed with a FACScan cytofluorometer (FACS Canto BD), and data were analyzed with FlowJo software.

RNA analysis
Expression of mRNA was examined by qRT-PCR using a 7900 Fast Real Time thermocycler and Fast Sybr Green assays (Applied Biosystems). Relative mRNA expression was normalized to Gapdh and Actb mRNA. The primers used were as follows: Actb (F: GGCTGTATTCCCCTCCATCG; R: CCAG

Measurement of hepatic triglycerides
Lipids were extracted from 25 mg of liver in isopropanol (50 mg/mL) and centrifuged (15 min 9500 xg 4˚C). Triglycerides were detected in the supernatant (Sigma-Aldrich).

Histology
Tissue samples were fixed in 10% formalin for 48 hr, dehydrated, and embedded in paraffin. Sections (5 mm) were cut and stained with hematoxylin and eosin (Sigma-Aldrich and Thermo Scientific). Sections (8 mm) from frozen tissue and embedded in OCT compound (Tissue-Tek) were stained with Oil Red O (American Master Tech Scientific). Sections were examined in Leica DM2500 microscope using 20x objective.
NE measurement 20 mL of PBS prefunded livers were crushed with a syringe plunger, resuspended in 4 mL of PBS/ EDTA 5 mM/0.5% FBS and filtered (70 mm). Cell suspension was centrifuged at 1800 rpm 5 min and the supernatant was filtered (22 mm). Supernatants were concentrated using Amicon Ultra centrifugal filters (Sigma-Aldrich). NE levels were determined with Mouse Neutrophil Elastase ELISA kit (R and D system).

Quantification and statistical analysis
All data are expressed as means ± SEM. For comparisons between two groups, the Student's t-test was applied. For data with more than two data sets, we used one-way ANOVA coupled with Turkey's multigroup test. When variances were unequal, Welch's test or Kruskal-Wallis test coupled with Dunn's multiple comparison test were applied, respectively. Multiple group comparisons in the rhythmicity of neutrophil infiltration were analyzed with two-way ANOVA followed by Fisher's post hoc test. Significance was determined as a 2-sided p < 0.05. All statistical analyses were conducted in GraphPad Prism software. Statistical details were indicated in the figure legends. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.