Fei Teng

Jeremy Goc

Lei Zhou

Coco Chu

Manish A Shah

Gérard Eberl

Gregory F Sonnenberg
email: gfsonnenberg@med.cornell.edu

A circadian clock is essential for homeostasis of group 3 innate lymphoid cells in the gut

Group 3 innate lymphoid cells (ILC3) critically orchestrate host-microbe interactions in the healthy mammalian intestine and become substantially impaired in the context of inflammatory bowel disease (IBD). However, the molecular pathways controlling the homeostasis of ILC3 remain incompletely defined. Here, we identify that intestinal ILC3 are highly enriched in expression of genes involved in the circadian clock and exhibit diurnal oscillations of these pathways in response to light cues. Classical ILC3 effector functions also exhibited diurnal oscillations and lineage-specific deletion of BMAL1, a master regulator of the circadian clock, resulted in dramatically reduced ILC3s selectively in the intestine. BMAL1-deficient ILC3 exhibit impaired expression of Nr1d1 and Per3, hyper-activation of RORγt-dependent target genes, and elevated pro-apoptotic pathways. Depletion of the microbiota with antibiotics partially reduced the hyper-activation of BMAL1-deficient ILC3 and restored cellular homeostasis in the intestine. Finally, ILC3 isolated from the inflamed intestine of patients with IBD exhibit substantial alterations in expression of several circadian-related genes. Our results collectively define that circadian regulation is essential for the homeostasis of ILC3 in the presence of a complex intestinal microbiota, and further that this pathway is disrupted in the context of IBD.

INTRODUCTION

The mammalian gastrointestinal tract is continuously exposed to both pathogenic and commensal microbes, and the immune system must carefully regulate immunity, inflammation and tolerance at this site in order to maintain tissue homeostasis [START_REF] Turner | Intestinal mucosal barrier function in health and disease[END_REF][START_REF] Belkaid | Homeostatic Immunity and the Microbiota[END_REF][START_REF] Hooper | Interactions between the microbiota and the immune system[END_REF]. Innate lymphoid cells (ILCs) have emerged as critical regulators of these processes due to their enrichment and tissue residency in the intestine, ability to rapidly respond to microbial stimuli, and multiple protective, anti-microbial or immune-modulatory functions [START_REF] Artis | The biology of innate lymphoid cells[END_REF][START_REF] Eberl | Innate lymphoid cells. Innate lymphoid cells: a new paradigm in immunology[END_REF][START_REF] Klose | Innate lymphoid cells as regulators of immunity, inflammation and tissue homeostasis[END_REF][START_REF] Wojno | Emerging concepts and future challenges in innate lymphoid cell biology[END_REF][START_REF] Vivier | Innate Lymphoid Cells: 10 Years On[END_REF]. In particular, group 3 ILCs (ILC3) play numerous roles in regulating intestinal health through production of IL-22, as well as their ability to control adaptive immune responses to dietary antigens or the intestinal microbiota [START_REF] Sonnenberg | Border patrol: regulation of immunity, inflammation and tissue homeostasis at barrier surfaces by IL-22[END_REF][START_REF] Sonnenberg | CD4(+) lymphoid tissue-inducer cells promote innate immunity in the gut[END_REF][START_REF] Spencer | Adaptation of innate lymphoid cells to a micronutrient deficiency promotes type 2 barrier immunity[END_REF][START_REF] Van De Pavert | Maternal retinoids control type 3 innate lymphoid cells and set the offspring immunity[END_REF][START_REF] Hepworth | Innate lymphoid cells regulate CD4+ T-cell responses to intestinal commensal bacteria[END_REF][START_REF] Hepworth | Immune tolerance. Group 3 innate lymphoid cells mediate intestinal selection of commensal bacteria-specific CD4(+) T cells[END_REF][START_REF] Zhou | Innate lymphoid cells support regulatory T cells in the intestine through interleukin-2[END_REF][START_REF] Sonnenberg | Functional interactions between innate lymphoid cells and adaptive immunity[END_REF]. Consistent with this, experimental manipulation of ILC3 in mice frequently results in spontaneous inflammation, microbial dysbiosis, or increased susceptibility to tissue damage and infection in the intestine [START_REF] Hepworth | Innate lymphoid cells regulate CD4+ T-cell responses to intestinal commensal bacteria[END_REF][START_REF] Hepworth | Immune tolerance. Group 3 innate lymphoid cells mediate intestinal selection of commensal bacteria-specific CD4(+) T cells[END_REF][START_REF] Goto | Innate lymphoid cells regulate intestinal epithelial cell glycosylation[END_REF][START_REF] Sonnenberg | Innate lymphoid cells promote anatomical containment of lymphoid-resident commensal bacteria[END_REF]. Furthermore, humans with chronic intestinal inflammation or infections exhibit substantially impaired ILC3 responses [START_REF] Hepworth | Immune tolerance. Group 3 innate lymphoid cells mediate intestinal selection of commensal bacteria-specific CD4(+) T cells[END_REF][START_REF] Zhou | Innate lymphoid cells support regulatory T cells in the intestine through interleukin-2[END_REF][START_REF] Forkel | Dysregulation of Group 3 Innate Lymphoid Cells in the Pathogenesis of Inflammatory Bowel Disease[END_REF][START_REF] Geremia | Innate Lymphoid Cells in Intestinal Inflammation[END_REF][START_REF] Kloverpris | Innate Lymphoid Cells Are Depleted Irreversibly during Acute HIV-1 Infection in the Absence of Viral Suppression[END_REF][START_REF] Bernink | Human type 1 innate lymphoid cells accumulate in inflamed mucosal tissues[END_REF], implicating a critical role for these cells in regulating human health.

Although many advances have shed light on how ILC3 maintain intestinal health and become impaired in human diseases, there is a limited understanding of the cellular and molecular pathways that are essential for ILC3 homeostasis in the gut. One identified factor is the microbiota, which can support the development of T-bet + ILC3 responses in the intestine [START_REF] Kiss | Natural aryl hydrocarbon receptor ligands control organogenesis of intestinal lymphoid follicles[END_REF][START_REF] Klose | A T-bet gradient controls the fate and function of CCR6-RORgammat+ innate lymphoid cells[END_REF][START_REF] Vonarbourg | Regulated expression of nuclear receptor RORgammat confers distinct functional fates to NK cell receptor-expressing RORgammat(+) innate lymphocytes[END_REF]. Intriguingly, recent studies have demonstrated that the intestinal microbiota exhibits diurnal oscillation in luminal abundance, epithelial attachment and metabolism [START_REF] Thaiss | Transkingdom control of microbiota diurnal oscillations promotes metabolic homeostasis[END_REF][START_REF] Thaiss | Microbiota Diurnal Rhythmicity Programs Host Transcriptome Oscillations[END_REF][START_REF] Leone | Effects of diurnal variation of gut microbes and high-fat feeding on host circadian clock function and metabolism[END_REF][START_REF] Liang | Rhythmicity of the intestinal microbiota is regulated by gender and the host circadian clock[END_REF]. This circadian cycle of the microbiota also influences the transcriptomic and epigenomic patterns of host cells both local and distal to the gut [START_REF] Thaiss | Microbiota Diurnal Rhythmicity Programs Host Transcriptome Oscillations[END_REF][START_REF] Weger | The Mouse Microbiome Is Required for Sex-Specific Diurnal Rhythms of Gene Expression and Metabolism[END_REF]. Mammalian hosts also exhibit diurnal oscillations or a circadian clock. These pathways are regulated by, or consist of, complex neuronal signaling from the synchronization of environmental light cues by the suprachiasmatic nuclei (SCN) in the hypothalamus and release of hormones or metabolites to the periphery [START_REF] Thaiss | Microbiota Diurnal Rhythmicity Programs Host Transcriptome Oscillations[END_REF][START_REF] Zhang | A circadian gene expression atlas in mammals: implications for biology and medicine[END_REF][START_REF] Buijs | The Circadian System: A Regulatory Feedback Network of Periphery and Brain[END_REF][START_REF] Dumbell | Circadian Clocks, Stress, and Immunity[END_REF]. This can in turn influence multiple immune responses and non-hematopoietic cells through entrainment and a cell autonomous circadian clock [START_REF] Scheiermann | Circadian control of the immune system[END_REF][START_REF] Scheiermann | Clocking in to immunity[END_REF]. In the intestine, it was recently identified that intestinal epithelial cells (IEC) exhibit diurnal oscillation in transcription patterns that are required to orchestrate normal lipid metabolism, and this was in part regulated by the microbiota, IL-22 and IEC-intrinsic expression of NFIL3 [START_REF] Wang | The intestinal microbiota regulates body composition through NFIL3 and the circadian clock[END_REF]. However, it remains unclear whether ILC3 exhibit circadian regulation and whether this is important for their cellular homeostasis.

Here we provide the first report that ILC3 exhibit diurnal oscillations in expression of genes related to the circadian clock and classical effector functions, and further, that ILC3-specific BMAL1 is required for cellular homeostasis in the presence of a diverse microbiota. Importantly we identify that ILC3 in the inflamed intestine of IBD patients exhibit alterations in circadian gene expression. These findings reveal that circadian regulation critically orchestrates ILC3 homeostasis in the gut, and suggest that disruption in this pathway drives the impaired ILC3 responses in the inflamed intestine of IBD patients.

RESULTS

To begin to examine whether ILC3 exhibit circadian regulation, we sort-purified ILC3 from the small intestinal lamina propria (SI-LP) of healthy mice and profiled them with RNA sequencing. Strikingly, in comparison to the related GATA3 + group 2 ILC (ILC2) population, RORγt + ILC3 exhibited enrichment in multiple transcripts associated with a cell-intrinsic circadian clock, including Per1, Per2, Per3, Cry1, Cry2, Nr1d1 and Nfil3 (Fig. 1A). To verify these findings, we sort-purified several immune cell subsets from the SI-LP of C57BL/6 mice at Zeitgeber time (ZT) 6 (Fig. S1). Quantitative PCR (qPCR) analyses confirmed that among major immune cell subsets in the intestine, ILC3 exhibited significantly higher expression of key circadian genes, including Nr1d1, Per1, Cry2, Nfil3 and Arntl (Fig. 1B). We next examined whether ILC3 express these genes in a diurnal manner by sort-purifying SI-LP ILC3 at 4 time points during a 24-hour cycle. We observed by qPCR analyses that ILC3 express Nr1d1, Per1, Per2, Per3 and Cry2 in a diurnal oscillation pattern (Fig. 1C). Diurnal oscillations of peripheral cell types can be entrained by the suprachiasmatic nuclei (SCN) in the hypothalamus in response to light cues [START_REF] Thaiss | Microbiota Diurnal Rhythmicity Programs Host Transcriptome Oscillations[END_REF][START_REF] Zhang | A circadian gene expression atlas in mammals: implications for biology and medicine[END_REF][START_REF] Buijs | The Circadian System: A Regulatory Feedback Network of Periphery and Brain[END_REF][START_REF] Dumbell | Circadian Clocks, Stress, and Immunity[END_REF]. Consistent with this, a 9-hour phase shift in the light-dark cycle resulted in a comparable shift in the diurnal expression patterns of Nr1d1, Per1, Per2 and Cry2 (Fig. 1D). In contrast, depletion of a diverse microbiota with antibiotics (Abx) modestly impacted the diurnal oscillations of these circadian genes in ILC3 (Fig. S2A). Thus, our data indicate that intestinal ILC3 exhibit a cell-intrinsic circadian clock that is regulated in part by light cues.

A role for the circadian clock has been implicated in the function of multiple other immune cell types [START_REF] Scheiermann | Clocking in to immunity[END_REF]. Therefore, we next examined whether the classical effector functions of ILC3 exhibit circadian regulation. ILC3 are characterized by expression of the master regulator transcription factor RORγt and high expression of the effector cytokines IL-17 and IL-22 [START_REF] Sonnenberg | CD4(+) lymphoid tissue-inducer cells promote innate immunity in the gut[END_REF][START_REF] Sawa | Lineage relationship analysis of RORgammat+ innate lymphoid cells[END_REF]. We observed that both Rorc, Il17a, and Il22 transcripts exhibited diurnal oscillations within a 24-hour cycle (Fig. 2A). We were also able to isolate differential numbers of ILC3s from the intestine in a diurnal manner, observed oscillations of Csf2 expression in ILC3, and found a diurnal expression of Reg3b in intestinal epithelial cells (Fig. S2B-D). We verified diurnal oscillation patterns in intestinal ILC3s at the protein level by the staining intensity of RORγt (Fig. 2B). To verify oscillations in IL-22, we developed a novel IL-22 reporter mouse to permit the analyses of IL-22 production without the need for ex vivo re-stimulation (Fig. S3A) and validated that the GFP signal could robustly represent IL-22 protein production by ILC3 (Fig. S3B,C). Consistent with the oscillations of Il22 transcript, we observed significant changes in IL-22-eGFP levels between ZT6 and ZT18 in intestinal ILC3 from the reporter mice (Fig. 2C). The diurnal oscillations of Rorc and Il22 transcripts were regulated by both light cues and microbiota-derived signals (Fig. 2D, E andS2E, F). Taken together, these data demonstrate that major ILC3 regulators and effector functions also exhibit diurnal oscillations across a 24-hour cycle.

We next examined the functional significance of these diurnal oscillation patterns by generating mice with an ILC3-specific deletion in BMAL1, a master regulator of the circadian cycle encoded by Arntl [START_REF] Curtis | Circadian clock proteins and immunity[END_REF]. This was accomplished by crossing Rorc cre mice [START_REF] Sawa | Lineage relationship analysis of RORgammat+ innate lymphoid cells[END_REF] with BMAL1 fl/fl mice [START_REF] Storch | Intrinsic circadian clock of the mammalian retina: importance for retinal processing of visual information[END_REF], to generate BMAL1 ΔRorc mice that exhibit an efficient loss of BMAL1 protein in intestinal ILC3 (Fig. 3A). Although RORγt is also expressed in double positive thymocytes and maintained in Th17 cells, we did not observe a loss of BMAL1 protein in intestinal Th17 cells in these mice (Fig. 3A), suggesting a selective targeting of ILC3. We found ILC3 from BMAL1 ΔRorc mice exhibited significantly altered expression of RORγt and NFIL3 protein (Fig. 3B), indicating a functional impairment in this population. The lack of ILC3-intrinsic BMAL1 also led to significant reductions in both the frequency and cell number of ILC3 in the small intestine (Fig. 3C). This is in contrast to a recent study demonstrating that intestinal Th17 cells, an adaptive counterpart of ILC3, are modestly impacted by BMAL1 deletion [START_REF] Hemmers | The Cell-Intrinsic Circadian Clock Is Dispensable for Lymphocyte Differentiation and Function[END_REF]. Interestingly, we found a preferential requirement of BMAL1 in the T-bet + subset of ILC3 (Fig. 3D), a population that requires the microbiota for development [START_REF] Kiss | Natural aryl hydrocarbon receptor ligands control organogenesis of intestinal lymphoid follicles[END_REF][START_REF] Klose | A T-bet gradient controls the fate and function of CCR6-RORgammat+ innate lymphoid cells[END_REF][START_REF] Vonarbourg | Regulated expression of nuclear receptor RORgammat confers distinct functional fates to NK cell receptor-expressing RORgammat(+) innate lymphocytes[END_REF]. This was selective to small intestinal ILC3, as the cellularity of intestinal ILC2 and Th17 cells, along with ILC3 in lymphoid tissues, remained comparable to littermate controls (Fig. S4A-C). There were also comparable Peyer's patches (PPs) and myeloid cell-derived cytokines in both groups of mice, and there was no impact on the homeostasis of ILC3 following selective deletion of BMAL1 in T cells (Fig. S4D-F). The frequency and total numbers of ILC3 in the large intestine were comparable in the context of cell-intrinsic BMAL1 deletion, but significant reductions were observed in the T-bet + ILC3 subset (Fig. S5A,B). Littermate control and BMAL1 ΔRorc mice exhibited comparable susceptibility to dextran sodium-induced intestinal damage and inflammation (Fig. S5C,D), which is consistent with the potential redundancy of T-bet + ILC3 in this model [START_REF] Song | Unique and redundant functions of NKp46+ ILC3s in models of intestinal inflammation[END_REF]. Collectively, these data critically demonstrate that the master regulator of circadian cycles, BMAL1, is essential for the homeostasis of ILC3 selectively within the intestine.

To define how BMAL1 regulates ILC3 cellular homeostasis, we performed RNA-sequencing on sort-purified populations from the small intestine of littermate control and BMAL1 ΔRorc mice. Unbiased analyses revealed significant differences in transcriptional signatures, which were driven in part by altered expression of genes associated with circadian-regulation, RORγt-mediated transcription, and apoptosis pathways (Fig. 4A,B). Specifically, BMAL1deficiency in ILC3 resulted in significantly reduced expression on key circadian-associated genes Nr1d1, Nr1d2, and Per3, while Cry1 was significantly increased and other circadianrelated genes remained unchanged (Fig. 4C). NR1D1 (also known as Rev-erbα) is a transcriptional repressor that competes for binding to RORγt DNA consensus sequences [START_REF] Kojetin | REV-ERB and ROR nuclear receptors as drug targets[END_REF][START_REF] Amir | REV-ERBalpha Regulates TH17 Cell Development and Autoimmunity[END_REF]. Consistent with this, BMAL1-deificient ILC3 exhibited hyper-activation of RORγt-dependent target genes Il17a, Il17f and Il22, as well as the pro-apoptotic pathways Bcl2l11 (encoding Bim) and Bax (Fig. 4C). We verified that BMAL1-deficient ILC3 exhibited significantly impaired expression of Nr1d1 and Per3 at both ZT6 and ZT18, while Per1 and Cry2 were comparable at these time points, and Il22 was significantly increased at ZT6, relative to littermate controls (Fig. 4D,E). Expression of Reg3b in the intestinal epithelium was comparable between both groups of mice, which is likely the result of significantly elevated frequencies of IL-22-and IL-17A-producing ILC3 but reduced total numbers (Fig. 4F, G andS6A). Mice lacking ILC3-specific BMAL1 also exhibited comparable frequencies of proliferating Ki-67 + ILC3 but significantly increased levels of Bim + ILC3 relative to littermate controls (Fig 4 .H,I). The reduced frequencies of ILC3 and increased levels of Bim + ILC3s were observed as early as 4 weeks of age (Fig. S6B-D). These results collectively demonstrate that ILC3-specific BMAL1 critically promotes expression of several circadian-associated genes (Nr1d1 and Per3), limits hyper-activation of RORγt-dependent target genes (Il17a, Il17f and Il22), and prevents elevated expression of pro-apoptotic pathways (Bim and Bax).

There is a dynamic and reciprocal regulation of ILC3 with intestinal microbiota, which promotes expression of RORγt and its associated target genes [START_REF] Sonnenberg | Innate lymphoid cells promote anatomical containment of lymphoid-resident commensal bacteria[END_REF][START_REF] Mortha | Microbiota-dependent crosstalk between macrophages and ILC3 promotes intestinal homeostasis[END_REF][START_REF] Zhou | Essential immunologic orchestrators of intestinal homeostasis[END_REF][START_REF] Sonnenberg | Innate lymphoid cells in the initiation, regulation and resolution of inflammation[END_REF]. Given our new findings and previous reports that the microbiota exhibit diurnal oscillations [START_REF] Thaiss | Transkingdom control of microbiota diurnal oscillations promotes metabolic homeostasis[END_REF][START_REF] Thaiss | Microbiota Diurnal Rhythmicity Programs Host Transcriptome Oscillations[END_REF][START_REF] Leone | Effects of diurnal variation of gut microbes and high-fat feeding on host circadian clock function and metabolism[END_REF][START_REF] Liang | Rhythmicity of the intestinal microbiota is regulated by gender and the host circadian clock[END_REF], we next examined whether the microbiota is driving the observed alterations of ILC3 homeostasis following loss of BMAL1. This was accomplished by administering antibiotics to BMAL1 ΔRorc mice. After two weeks of Abx administration, we observed comparable protein levels of RORγt, but significantly reduced levels of IL-17A and IL-22 in ILC3 from Abx treated BMAL1 ΔRorc mice relative to non-treated BMAL1 ΔRorc mice (Fig. 5A). Strikingly, Abx-treatment also resulted in a partial restoration of ILC3 frequencies and a complete restoration of ILC3 numbers in BMAL1 ΔRorc mice (Fig. 5B). These results demonstrate that circadian regulation is essential for orchestrating homeostasis between ILC3 and the microbiota, and implicates that hyper-activation of RORγt is associated with the reduction of intestinal ILC3 in the context of BMAL1-deficiency. In support of this, we identified that small molecule inhibition of RORγt in purified ILC3 cultures was sufficient to reduce expression of BMAL1, as well as significantly reduce Bim + ILC3 in the context of simulation with pro-inflammatory cytokines that are typically elicited by the microbiota (Fig. 5C,D). Thus, hyper-activation and loss of intestinal ILC3 in the context of cellintrinsic BMAL deletion is driven in part by the microbiota.

IBD is an inflammatory disease of humans that is accompanied with microbial dysbiosis and disruption of intestinal ILC3 responses [START_REF] Hepworth | Immune tolerance. Group 3 innate lymphoid cells mediate intestinal selection of commensal bacteria-specific CD4(+) T cells[END_REF][START_REF] Zhou | Innate lymphoid cells support regulatory T cells in the intestine through interleukin-2[END_REF][START_REF] Bernink | Human type 1 innate lymphoid cells accumulate in inflamed mucosal tissues[END_REF]. To investigate for a contribution of circadian regulation, we sort purified ILC3 from the inflamed or non-inflamed regions of intestinal resections from IBD patients. We observed significantly reduced frequencies of ILC3 from the inflamed versus non-inflamed intestine of IBD patients, and strikingly, this correlated with significantly altered expression of the circadian-related genes NR1D1, PER3 and NFIL3 (Fig. 6A,B), while expression of ARNTL and CRY1 were not substantially altered. These results suggest that in the context of chronic intestinal inflammation, human ILC3 exhibit alterations in circadian gene expression, which may contribute to the altered homeostasis of these protective cell types.

DISCUSSION

Our results define that cell-intrinsic circadian regulation is critically required for the homeostasis of ILC3 in the intestine. The mammalian circadian clock plays important roles in regulating multiple immune cell functions [START_REF] Scheiermann | Circadian control of the immune system[END_REF][START_REF] Scheiermann | Clocking in to immunity[END_REF][START_REF] Tognini | Circadian Coordination of Antimicrobial Responses[END_REF], recent studies have shed light on how circadian clock influences both innate and adaptive counterparts of immunity, as well as non-immune cells to properly respond to environmental stimuli and maintain homeostasis [START_REF] Wang | The intestinal microbiota regulates body composition through NFIL3 and the circadian clock[END_REF][START_REF] Yu | TH17 cell differentiation is regulated by the circadian clock[END_REF][START_REF] Allen | Desynchronization of the molecular clock contributes to the heterogeneity of the inflammatory response[END_REF]. However, very little is known about the circadian clock and ILC3. An important earlier study demonstrated that ILC2 exhibit circadian regulation and that this was important to support eosinophil homeostasis [START_REF] Nussbaum | Type 2 innate lymphoid cells control eosinophil homeostasis[END_REF]. Here we provide the first evidence that intestinal ILC3 require a circadian clock to better sense and interact with the microbiota, adding another layer of complexity in the dialogue between ILCs and microbes (Fig. S8). This may be the result of coordinating diurnal cycles to facilitate normal ILC3 responses and fine-tune the function of RORγt. In the context of IBD patients, we identified that ILC3 in the inflamed intestine exhibit altered circadian gene expression. This is consistent with prior reports that characterized altered circadian gene expression in whole intestinal biopsies from IBD patients [START_REF] Liu | Bidirectional Regulation of Circadian Disturbance and Inflammation in Inflammatory Bowel Disease[END_REF]. Given that our findings come from the same patient, it suggests that the altered expression is not being driven by body wide disruptions in circadian cycles or entrainment, but rather the result of chronic inflammation or microbial dysbiosis. Inflammatory disruption of circadian cycles has been reported in multiple other cell types [START_REF] Leone | Role of proinflammatory cytokines on lipopolysaccharide-induced phase shifts in locomotor activity circadian rhythm[END_REF][START_REF] Cavadini | TNF-alpha suppresses the expression of clock genes by interfering with E-boxmediated transcription[END_REF][START_REF] Westfall | Time-dependent effects of localized inflammation on peripheral clock gene expression in rats[END_REF]. Moreover, it has become increasingly appreciated that there are certain signatures of immune responses and therapies that exert day and night differences (termed as chronoimmunotherapy), and our understanding of this is already being applied at different levels in diseases such a COPD and rheumatoid arthritis [START_REF] Buttgereit | Efficacy of modified-release versus standard prednisone to reduce duration of morning stiffness of the joints in rheumatoid arthritis (CAPRA-1): a double-blind, randomised controlled trial[END_REF][START_REF] Canonica | Characterization of circadian COPD symptoms by phenotype: Methodology of the STORICO observational study[END_REF]. Therefore, our critical finding that ILC3 in the inflamed intestine of IBD patients exhibit altered circadian gene expression suggests that this is an important pathway in human health and disease, and may hold a key for developing novel strategies to boost ILC3 responses in the context of impaired intestinal homeostasis or microbial dysbiosis.

MATERIALS AND METHODS

Study design

The objective of this study was to interrogate the role of circadian regulation on ILC3 homeostasis in the gut. To do this, we employed a combination of ex vivo and in vivo assays with both mouse and human samples. We designed and performed the experiments mainly in the fields of cellular immunology and molecular biology. The number of replicates for each experiment is indicated in the figure legends.

Mice

Wild-type, BMAL1 fl/fl and Cd4 cre mice on a C57BL/6 background were purchased from the Jackson Laboratory. C57BL/6 Rorc cre mice and Rorc(γt)-Gfp TG mice were provided by G. Eberl. BMAL1 ΔRorc mice were generated by crossing Rorc cre mice with BMAL1 fl/fl mice and BMAL1 ΔCd4 mice were generated by crossing Cd4 cre mice with BMAL1 fl/fl mice. IL-22-eGFP mice were generated in collaboration with Cyagen US Inc by modifying a Bacterial Artificial Chromosome as noted in Figure S3A and injection into fertilized C57BL/6 embryos. All mice were bred and maintained in specific pathogen-free facilities under a standard 24 hour light:dark cycle (light on for 12 hours from 6am to 6pm as Zeitgeber times [ZT] 0-12) at Weill Cornell Medicine). In one noted experimental set mice were housed with a 9-hour advanced light:dark cycle, and the light was set on from 9pm to 9am. Mice were fed ad libitum. Sex-and age-matched littermates were used as controls in all experiments and mice were between 7 and 11 weeks of age where used for all experiments unless otherwise indicated. Mice were sacrificed at ZT5 to ZT6 unless otherwise indicated. All animal experiments were approved and are in accordance with the Institutional Animal Care and Use Committee guidelines at Weill Cornell Medicine.

Isolation of cells from the intestinal epithelium and lamina propria of mice and humans

Mouse intestines were removed, cleaned from remaining fat tissue and washed in ice-cold PBS (Corning). Peyer's patches on the small intestine were identified and completely eliminated. Intestines were opened longitudinally and washed in ice-cold PBS. Afterwards, mucus was gently removed by forceps and intestines were cut into approximately 0.5 cm sections. Dissociation of epithelial cells was performed by incubation on a shaker in HBSS (Sigma-Aldrich) containing 5 mM EDTA (Thermo Fisher Scientific), 1mM DTT (Sigma-Aldrich) and 2% heat inactivated FBS two times for 20 min each at 37 °C. After each incubation, samples were vortexed, after the 2 nd incubation, the epithelial fraction was washed by ice-cold PBS and proceeded with qPCR analysis. The remaining samples were then washed by cold PBS and enzymatic digestion was performed using dispase (0.4 U/ml; Thermo Fisher Scientific), collagenase III (1 mg/ml; Worthington) and DNase I (20 μg/ml; Sigma-Aldrich) in 10% FBS DMEM (Corning) on a shaker for 45 min at 37 °C. Leukocytes were further enriched by a 40/80% Percoll gradient centrifugation (GE Healthcare).

Surgical resection samples from IBD patients (see Table S1) were obtained through Institutional Review Board approved protocols from the Centers for Advanced Digestive Care at Weill Cornell Medicine following informed consent. Single cell suspensions from intestinal tissues were obtained by incubating tissues for 30 min at 37°C with shaking in stripping buffer (1 mM EDTA, 1 mM DTT and 5% FCS) to remove the epithelial layer. Supernatants were then discarded. Tissues were then mechanically dissociated with a sterile scalpel. The lamina propria fraction was obtained by incubating the dissociated tissues for 1 hour at 37°C with shaking in 2 mg/ml collagenase D (Roche), 0.1 mg/ml DNase I (Sigma) and 1 mg/ml of Trypsin Inhibitor (Gibco) digestion solution. Remaining tissues were then filtered through a 70 μm cell strainer. All cells were then viably cryopreserved in 90% FBS and 10% DMSO for side-by-side analysis at a later time point. Following thawing and filtered through a 70μm cell strainer, cells were stained with antibodies for flow cytometry acquisition.

Flow cytometry and cell sorting

Single cell suspensions were incubated on ice with conjugated antibodies in PBS containing 2% FBS and 1mM EDTA. Dead cells were excluded with Fixable Aqua Dead Cell Stain (Thermo Fisher Scientific). The staining antibodies for flow cytometry were purchased from Thermo Fisher Scientific, Biolegend, BD Biosciences or Cell Signaling Technology. For mouse cell-surface staining: CD45 (30-F11), CD3ε (145-2C11), CD5 (53-7.3), CD8α (53-6.7), NK1.1 (PK136), CD11c (N418), CD11b (M1/70), B220 (RA3-6B2), CD19 (eBio1D3), TCRβ (H57-597), CD64 (X54-5/7.1), MHC-II (M5/114.15.2), CD90.2 (30-H12), CD127 (A7R34), CD27 (LG.7F9), CD4 (GK1.5 or RM4-5), KLRG1 (2F1). For mouse intracellular staining: T-bet (4B10), GATA3 (L50-823), RORγt (B2D or Q31-378), NFIL3 (S2M-E19), Ki-67 (SolA15), Bim (C34C5), IL-17A (eBio 17B7), IL-22 (IL22JOP). Intracellular staining of mouse BMAL1 was conducted by first staining polyclonal anti-BMAL1 antibody (Abcam) then further staining with fluorescent conjugated donkey antirabbit IgG antibody (BioLegend). Human samples were stained for CD3 (UCHT1), CD5 (UCHT2), CD11b (CBRM1/5), CD11c (3.9), CD19 (HIB19), CD45 (HI30), CD117 (104D2), CD127 (A019D5), FcεR1 (AER-37) and CRTH2 (BM16).

For intracellular transcription factor or cytokine staining, cells were stained for surface markers, followed by fixation and permeabilization before nuclear factor staining according to the manufacturer's protocol (Foxp3 staining buffer set from Thermo Fisher Scientific). For intracellular cytokine staining, cells were incubated for 4 hours at 37 °C in complete medium (DMEM with 10% FBS, 10 mM HEPES, 1 mM sodium pyruvate, non-essential amino acids, 80 μM 2-mercaptoethanol, 100 U/ml penicillin and 100 μg/ml streptomycin, all from Gibco), supplied with phorbol 12-myristate 13-acetate (PMA) (50 ng/ml), ionomycin (750 ng/ml) and brefeldin A (10 μg/ml) (all from Sigma-Aldrich). All flow cytometry experiments were performed using a Fortessa flow cytometer and the FACS Diva software (BD Biosciences) and analyzed with FlowJo V10 software (TreeStar) or sort-purified by using FACSAria II cell sorter (BD Biosciences).

RNA sequencing analysis

ILC2 and ILC3 were sort-purified from small intestine of Rorc(γt)-Gfp TG mice or BMAL1 fl/fl and BMAL1 ΔRorc littermates. Sorted cells were used to prepare RNA-seq libraries by the Epigenomics Core at Weill Cornell Medicine using the Clontech SMARTer Ultra Low Input RNA Kit V4 (Clontech Laboratories). Sequencing was performed on an Illumina HiSeq 2500, yielding 50 bp single-end reads.

Raw sequencing reads were demultiplexed with Illumina CASAVA (v1.8.2). Adapters were trimmed from reads using FLEXBAR (v2.4) and reads were aligned to the NCBI GRCm38/ mm10 mouse genome using the STAR aligner (v2.5.2b) with default settings. Reads per gene were counted using Rsubread. Genes with at least 50 or more counts in at least 2 samples were tested for differential expression. Differential expression was assessed using DESeq2 version 1.22.2 with default parameters and with a false discovery rate (FDR) of 0.1. Principal component analysis (PCA) was performed after applying the DESeq2 varianceStabilizingTransformation function, using the 500 genes with highest variance.

Quantitative PCR

Intestinal epithelial cells or sort-purified ILC3 were lysed in Buffer RLT (QIAGEN) or single-cell lysis buffer (Clontech-Takara). RNA was extracted via RNeasy mini kits (QIAGEN) as per the manufacturer's instructions. Reverse transcription of RNA was performed using Maxima reverse transcription or SuperScript VILO according to the protocols provided by the manufacturer (Thermo Fisher Scientific). Real-time PCR was performed on cDNA using SYBR green chemistry (Applied Biosystems). Reactions were run on a real-time PCR system (ABI7500; Applied Biosystems). Samples were normalized to Actb2, Hprt1 or GAPDH and displayed as a fold change or relative values compared to controls (see Table S2 for detailed primer information).

In vivo administration of antibiotics 0.5 mg/mL of ampicillin (Santa Cruz) and 0.5 mg/mL gentamicin (Gemini BioProducts) were continuously administered via drinking water for 2 weeks. Mice were 7-8 weeks of age when antibiotic treatment started.

In vitro stimulation with GSK805

GSK805 (Small molecule RORγt inhibitor) was purchased from EMD Millipore at a purity ⩾ 95%. ILC3 were sort-purified from SI-LP of Rorc(γt)-Gfp TG mice and incubated for 6 hours at 37 °C with GSK805 or control vehicle in complete medium, before proceeding with qPCR analysis. Bulk isolated SI-LP cells from C57BL/6 mice were incubated for 4 hours at 37 °C with GSK805 or control vehicle in complete medium, supplied with recombinant mouse IL-1β, IL-6, IL-12, IL-23 (20ng/ml each, all from Thermo Fisher Scientific) and analyzed by flow cytometry.

DSS induced colitis

3.5 grams DSS (MP Biomedicals) was dissolved in 100 ml drinking water. 6-7 weeks old BMAL1 fl/fl and BMAL1 ΔRorc littermates were given ad libitum access to DSS containing water for 7 consecutive days, the water was changed to normal drinking water from day 8. The mice were sacrificed on day 13 for measuring colon length and histology analysis.

For histology, distal colonic tissues were fixed with 4% paraformaldehyde (bioWORLD), embedded in paraffin, and 5 mm sections were stained with haematoxylin and eosin (H&E) by IDEXX BioResearch. Images were acquired using Nikon Eclipse Ti microscope (Nikon).

Statistical analysis

P values of mouse data sets were determined by one-way ANOVA, unpaired or paired twotailed Student's t-test with 95% confidence interval. For human data, significance was determined by a paired two-tailed Students' t-test with 95% confidence interval. All A. Heatmap showing expression Z-scores of the indicated genes in ILC2 (CD45 + Lin -CD90.2 + CD127 + CD27 -KLRG1 + ) and ILC3 (CD45 + CD3ε -CD127 + RORγt GFP+ ) from SI-LPs of naïve Rorc(γt)-Gfp TG mice, as measured by RNA-sequencing (Lineage markers: CD3ε, CD5, CD8α, NK1.1, CD11c, CD11b, B220). B. qPCR analyses of indicated circadian genes from sort-purified T cells, B cells, dendritic cells (DC), macrophages (Mac), ILC2 and ILC3 from SI-LP of C57BL/6 mice sacrificed at ZT6. The expression of each target gene was normalized to Actb2 (n=7, pooled from 2 independent assays). C. qPCR analyses of diurnal expression patterns in indicated circadian genes on sort-purified ILC3 from SI-LP of C57BL/6 mice sacrificed at ZT0, ZT6, ZT12 and ZT18 within a 24-hour cycle. The expression of each target gene at each time point was first normalized to Actb2, then further normalized to the sample with lowest expression at ZT0 (n=4, representative of 2 independent assays). D. qPCR analyses of diurnal expression patterns in indicated circadian genes on sort-purified ILC3 from SI-LP of C57BL/6 mice housed in standard light:dark cycle (control) or mice housed in 9-hour advanced light:dark cycle (L:D phase advanced). Both groups of mice were sacrificed at ZT0, ZT6, ZT12 and ZT18 within a 24hour cycle. The expression of each target gene at each time point was first normalized to Actb2, then further normalized to the sample with lowest expression at ZT0 in the control group (n=6/group, pooled from 2 independent assays). Results are shown as the mean ± s.e.m. Statistics are calculated by one-way ANOVA. Mice were sacrificed at ZT6 and ZT18, cells were gated on live CD45 dim Lin -CD90.2 + CD127 + CD27 -KLRG1 -(n=10-11, pooled from 3 independent assays). D. qPCR analysis of Il22 expression on sort-purified ILC3 from SI-LP of C57BL/6 mice housed in standard light:dark cycle or mice housed in 9-hour advanced light:dark cycle. Both groups of mice were sacrificed at ZT0, ZT6, ZT12 and ZT18 within a 24-hour cycle. The relative gene expression was normalized as Fig. 1D (n=6/group, pooled from 2 independent assays). E. qPCR analysis of Il22 expression on sort-purified ILC3 from SI-LP of conventionally housed C57BL/6 mice or mice treated with Abx for 2 weeks. Both groups of mice were sacrificed at ZT0, ZT6, ZT12 and ZT18 within a 24-hour cycle. The gene expression at each time point was first normalized to Actb2, then further normalized to the sample with lowest expression at ZT0 in the control group (n=6/group, pooled from 2 independent assays). Results are shown as the mean ± s.e.m. Statistic is calculated by unpaired two-tailed Student's t-test.

  statistical tests were performed with Graph Pad Prism V8 software. Data are shown as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 1 .

 1 Figure 1. Intestinal ILC3 are enriched for, and exhibit diurnal oscillations in, circadian-related gene expression.

Figure 2 .

 2 Figure 2. ILC3-specific effector and regulatory pathways exhibit diurnal oscillations. A. qPCR analyses of Rorc, Il17a and Il22 expression on sort-purified ILC3 from SI-LP of C57BL/6 mice sacrificed at ZT0, ZT6, ZT12 and ZT18 within a 24-hour cycle. The expression of each target gene at each time point was first normalized to Actb2, then further normalized to the sample with lowest expression at ZT0 (n=4, representative of 2 independent assays). B. MFI of RORγt diurnal expression in ILC3 from large intestine lamina propria (LI-LP) of mice from Fig. 2A, (n=4, representative of 2 independent assays). C. Representative flow cytometry plots and bar graphs of frequency of IL-22-eGFP

Figure 3 .

 3 Figure 3. Cell-intrinsic BMAL1 is required for ILC3 homeostasis in the intestine. A. Representative histogram overlay of BMAL1 expression in SI-LP ILC3 and Th17 cells from BMAL1 fl/fl mice and BMAL1 ΔRorc mice. ILC3 were gated as live CD45 + Lin -CD90.2 + CD127 + GATA-3 -RORγt + , Th17 cells were gated as live CD45 + CD3ε + CD4 + RORγt + IL-17A + . B. Bar graphs of MFI of RORγt and NFIL3 in SI-LP ILC3 from BMAL1 fl/fl mice and BMAL1 ΔRorc mice (representative of 3 independent assays). C. Representative flow cytometry plots and bar graphs of frequency and cell numbers of SI-LP ILC3 from BMAL1 fl/fl mice and BMAL1 ΔRorc mice. Cells were gated on live CD45 + Lin

Figure 4 .

 4 Figure 4. BMAL1-deficiency results in circadian dysregulation, cytokine hyper-production and increased cell death in intestinal ILC3. A. Principle component analysis (PCA) of genome-wide transcriptional profiles of sortpurified ILC3 from SI-LP of BMAL1 fl/fl mice and BMAL1 ΔRorc mice, as measured by RNA-sequencing. n=3/group. B. Volcano plot of differential expression between BMAL1 fl/fl (positive log2(fold change (FC))) and BMAL1 ΔRorc (negative log2(FC)) groups. Differentially expressed genes (defined as False Discovery Rate (FDR) < 0.1) are shown in red. C. Heatmap showing expression Z-scores of the indicated genes in ILC3 from SI-LP of

Figure 5 .

 5 Figure 5. Microbiota drives the dysfunction of ILC3 in the absence of BMAL1. A. Bar graphs of MFI of RORγt in ILC3, frequency of IL-17 + and IL-22 + ILC3 from SI-LP of Abx treated or control BMAL1 fl/fl mice and BMAL1 ΔRorc mice (representative of 3 independent assays). B. Representative flow cytometry plots and bar graphs of frequency and cell numbers of SI-LP ILC3 from Abx treated or control BMAL1 fl/fl mice and BMAL1 ΔRorc mice. Cells were gated as Fig. 3C (representative of 3 independent assays). C. qPCR analysis of Arntl expression on sort-purified SI-LP ILC3 from Rorc(γt)-Gfp TG mice treated with 2μM of the RORγt inhibitor GSK805 or vehicle control. The gene expression

Figure 6 .

 6 Figure 6. Circadian regulation of ILC3 is disrupted in IBD patients. A. Representative flow cytometry plots and bar graph of frequency of ILC3 from distal noninflamed versus matched inflamed surgical resection tissues of IBD patients. Cells were gated on live CD45 + Lin -CD127 + (Lineage markers: CD3, CD5, CD11b, CD11c, CD19, FcεR1). B. qPCR analyses of indicated circadian genes on sort-purified ILC3 from distal non-inflamed versus matched inflamed surgical resection tissues of IBD patients. The expression of each target gene was normalized to GAPDH. Statistics are calculated by paired two-tailed Student's t-test.

Sci Immunol. Author manuscript; available in PMC 2020 October 04.

ACKNOWLEDGEMENTS:

We thank members of the Sonnenberg Laboratory for discussions and critical reading of the manuscript. We would also like to thank the Epigenomics Core of Weill Cornell Medicine, Gregory G. Putzel, and the Center for Advanced Digestive Care (CADC).

Funding: Research in the Sonnenberg Laboratory is supported by the National Institutes of Health (R01AI143842, R01AI123368, R01AI145989 and U01AI095608), the NIAID Mucosal Immunology Studies Team (MIST), the Crohn's and Colitis Foundation of America, the Searle Scholars Program, the American Asthma Foundation Scholar Award, Pilot Project Funding from the Center for Advanced Digestive Care (CADC), an Investigators in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund, a Wade F.B. Thompson/Cancer Research Institute CLIP Investigator grant, the Meyer Cancer Center Collaborative Research Initiative, and the Jill Roberts Institute (JRI) for Research in IBD. Gregory F. Sonnenberg is a CRI Lloyd J. Old STAR. L.Z. and J.G. are supported by fellowships from the Crohn's and Colitis Foundation (608975 and 519428).

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.


Lassibille Gérard

Iredu

Teachers' Engagement at Work in a Developing Country

Using data from a survey conducted recently in Madagascar, this article analyses what teachers and school directors do when they are at work and how they manage the pedagogical process. The results show that in only 15% of the sample schools do all of the teachers and school directors consistently perform the tasks considered essential to their role. Engagement at work is found to be significantly lower among contract teachers than among civil service teachers, and the ability of teachers to manage the pedagogical process does not improve with accumulated experience. Other noteworthy features in the results are that the principal is the key agent in the school and that leadership is vitally important in developing effective schools.

Introduction

Education economists have spent considerable effort examining what factors affect academic achievement. While there is a large literature on the importance of financial resources in determining educational outcomes [START_REF] Hanushek | Assessing the Effects of School Resources on Student Performance: An Update[END_REF][START_REF] Filmer | What Education Production Functions Really Show: A Positive Theory of Education Expenditures[END_REF], researchers have paid considerably less attention to the management of the pedagogical process and its impact on schooling outcomes. That school management and teachers' engagement at work need more attention is suggested by the near absence of a link between the resources that a school receives and the schooling outcomes it delivers (see, for example, [START_REF] Lassibille | Student Learning in Public and Private Primary Schools in Madagascar[END_REF][START_REF] Banerjee | Promoting School Participation in Rural Rajasthan: Results from Some Prospective Trials[END_REF][START_REF] Duflo | Peer Effects, Pupil-Teacher Ratios, and Teacher Incentives[END_REF]Glewwe et al., 2009b). This paper explicitly considers these rather neglected aspects of educational achievement. It attempts to document and present information on how public primary school teachers in a developing country conduct their work and manage their classroom. It adds to the current base of knowledge on education quality and provides insightful lessons for the development of policies that seek to improve the management of primary schools in low-income countries. The data for the analysis reported in this paper pertain to a survey of some 300 schools with information on teachers and school heads, conducted in 2007 in the framework of a school management programme implemented in Madagascar (World Bank, 2010). By taking advantage of survey data of uncommon richness, the article analyses how principals and teachers in public primary schools handle tasks that educators deem essential to their role and examines the effects of select teacher characteristics and school demographics on engagement at work among school personnel. The findings of this paper can be summarised as follows. Many aspects of the pedagogical process are poorly managed, and tasks that are essential for student learning are neglected. For example, about 20% of teachers do not prepare daily lesson plans, school directors rarely follow up with their teaching staff on student performance, communication from teachers to parents on student learning is often perfunctory, pupil absences are rarely recorded and communicated to parents, and in only 15% of the sample schools do all of the teachers and school directors consistently perform the tasks considered essential by Malagasy educators. Beyond documenting teachers' behaviour at work, the paper evaluates the extent to which differences in teachers and school characteristics are associated with teachers' engagement at work. The type of employment contract held by teachers is found to be a potent influence of teachers' engagement at work. The empirical evidence shows that contract teachers are significantly less likely to execute conscientiously the tasks that are considered essential to their role compared with civil service teachers. This is an important result because Madagascar, like many others developing countries, has recently hired a large number of contract teachers who are paid much lower salaries than civil service teachers, and have fairly limited promotion opportunities within the education sector. When teacher pay is low, the incentives to generate additional income from secondary activity are generally high. In this regard, the results indicate that having a second job significantly affects work effort in the main job. Another noteworthy feature in the results is that school leadership has a significant impact on teachers' engagement at work and is vitally important in developing effective schools and in facilitating quality teaching and learning. The remainder of the paper is organised as follows. Section 2 briefly reviews the existing literature on teachers' engagement at work. Section 3 introduces the data. Section 4 provides empirical evidence on how school personnel conduct their work. Section 5 identifies predictors of teachers' engagement at work. The paper concludes in Section 6 with a discussion on the policy implications of our findings.

Literature review

Although the way teachers spend their time as workers is important from various policy perspectives, the literature has paid little attention to this issue. No large-scale study has examined how teachers conduct their work, although the early research by [START_REF] Hilsum | The Teachers' Day[END_REF] and [START_REF] Hilsum | The Secondary Teachers' Day: Report of the NFER Research Project[END_REF] maps out baseline data on the working days of junior and secondary school teachers in the UK. These influential studies were updated 20 years later by [START_REF] Campbell | Primary Teachers at Work[END_REF], who analyse the work of about 300 primary school teachers in England and Wales and describe the time they spent on teaching, preparation, grading, administration and professional development. By relating teachers' behaviour and conscientiousness to issues of school management and curriculum manageability, this emerging body of research has contributed to the understanding of teacher effectiveness. In the education and occupational psychology literature, several studies have focused on work engagement among teachers and the factors influencing teachers' engagement at work (see, for example, [START_REF] Rutter | Facilitating Teacher Engagement[END_REF][START_REF] Hakanen | Burnout and Work Engagement among Teachers[END_REF][START_REF] Kirkpatrick | To invest, coast or idle: second-stage teachers enact their job engagement[END_REF]. The majority of these works are qualitative case studies, with little statistical analysis. Work engagement is generally defined as a positive state of mind. A highly engaged employee is defined as one who is highly motivated, is committed to his job and believes that his job is meaningful and challenging (see, for example, [START_REF] Brown | A Meta-analysis and Review of Organizational Research on Job Involvement[END_REF]. Although this body of research does not focus directly on the way school personnel spend their time at work, certain behaviours, like spending extra time preparing classes or tutoring students, are obviously good indicators of work engagement. In the context of developing countries, a rapidly expanding literature documents teachers' absence and evaluates the effectiveness of interventions designed to reduce absenteeism (see, for example, [START_REF] Alcázar | Why Are Teachers Absent? Probing Service Delivery in Peruvian Primary Schools[END_REF][START_REF] Banerjee | Addressing Absence[END_REF][START_REF] Chaudhury | Missing in Action: Teacher and Health Worker Absence in Developing Countries[END_REF]Glewwe et al., 2009a). However, a search of published sources reveals a disappointingly small literature on what is really occurring in classrooms when teachers are present and on the way school personnel conduct their work. [START_REF] Basikin | Vigor, dedication, and absorption: work engagement among secondary school English teachers in Indonesia[END_REF] investigates work engagement among a sample of secondary school English teachers in Indonesia. Teachers' work engagement is measured on a three-factor scale consisting of nine items aiming to measure three specific dimensions of work engagement: effort (vigour), enthusiasm (dedication) and immersion in the work. The results indicate that work engagement among secondary school English teachers in Indonesia is generally high and that observable teacher characteristics are not significantly related to teachers' work engagement; unfortunately, the study does not address the impact on student learning of teacher engagement in teaching. During successive unannounced visits to a sample of public primary schools in India, [START_REF] Pandey | Public Participation, Teacher Accountability, and School Outcomes: Findings from Baseline Surveys in Three Indian States, Policy Research Working Paper[END_REF] measured engagement in teaching by identifying teachers who were teaching, writing on the board, supervising written work or teaching by rote. They find substantial variation in teacher effort within schools and a positive and significant correlation between engagement in teaching and student learning achievement. However, the gain in test score is small largely because teacher activity might not precisely measure real teacher effort. [START_REF] Adekanmbi | The Republic of The Gambia: Department of State for Basic and Secondary Education[END_REF] present results of a baseline survey administered in the context of an impact evaluation of two pilot programmes in the Republic of The Gambia. The authors consider whether teachers had a lesson plan or not. The data were collected during unannounced visits to public primary schools. The results indicate that more than half of the teachers did not have a lesson plan or could not show it when asked. This very partial attempt to describe teachers' work suggests that many teachers do not execute conscientiously the tasks for which they are responsible and that improving the management of work flow processes is needed in order to improve the performance and efficiency of the education system. This paper aims to investigate these aspects more deeply and to add to the emerging literature on teacher engagement at work in the context of developing countries. Taking advantage of a data set of uncommon richness, it analyses how school principals and teachers in public primary schools handle tasks that educators deem essential to their role and examines factors that influence their engagement at work.

The data

Data for the analysis come from surveys fielded with World Bank assistance in the framework of a school management programme implemented on an experimental basis by the Ministry of Education of Madagascar (the Amélioration de la Gestion de l'Education à Madagascar-AGEMAD; see World Bank, 2010). 1 Data on the behaviour of school personnel were collected in 2007 through a school questionnaire administered following a random schedule of unannounced visits to schools. The questionnaire generated detailed information on the pedagogical and administrative organisation of the school, on the personal characteristics and qualifications of the teachers and on teacher and student absenteeism. In addition to the standard teacher characteristics, the survey also gathered data on various aspects of school personnel behaviour and how teachers and principals do their job. This was done by asking each sampled teacher and school director to answer a series of questions about their administrative and pedagogical practices, 2 such as those controlling absence, preparing lesson plans, monitoring student learning and helping lagging pupils, communicating with parents and local school community. The data set used here includes information on 303 public primary schools and about 1,200 teachers; schools in the sample are located in eighty-four subdistricts distributed across fifteen school districts. In addition to the above, a survey of workflow processes was conducted at the end of the 2006 -07 school year. About 450 administrative and pedagogical work flow artefacts that provide a record of the work of school personnel over a full school year were collected from about 100 teachers in twenty randomly selected schools. This unique database reveals how teachers and school directors performed their duties throughout the school year, in particular regarding following actions: monitoring and following up on student absenteeism, preparing lesson plans, tracking progress in student learning, following up on teacher absenteeism. This information provides an independent external check on the data reported by school personnel to the survey enumerators. 3 As indicated below, there is a high degree of consistency between the two sources of information, a feature that strengthens confidence in the analytical results based on the reported data.

School personnel at work

What do teachers and school heads do when they are at work, and how do they manage the pedagogical process? The survey of work flow processes mentioned above offers particularly telling revelations in this regard (Table 1). The data show that pupil absenteeism is poorly monitored by teachers, with attendance taken, on average, only 13 days a month, and poorly supervised by school directors, with only one-third of the attendance records kept by teachers signed by school heads. Essential pedagogical tasks are often neglected: 14% of teachers do not use weekly lesson plans; teachers prepare daily lesson plans for a very small portion of the school year, covering an average of 34 days out of a total of 150 days; only 15% consistently prepare daily and weekly lesson plans; one-third of school heads never discuss with teachers their daily lesson plans. Students' academic progress is poorly monitored too: the results of tests and quizzes are badly recorded, communication from teachers to parents on student learning via student report cards is often perfunctory and pupil absences are rarely communicated to parents. School directors are seldom involved in following up on student performance: three-quarter do not discuss learning outcomes with teachers, according to the school survey data, and 80% of test results and student report cards are not signed by school heads. The same goes for teacher absences, which average nearly 10%-hardly a negligible figure 4 ; only 20% of school directors monitor teacher absences by taking daily attendance and a monthly summary of absences, with attendance taken, on average, only 3 months during the school year; more than 80% of them fail to report teacher absences to administrators at the subdistrict and district levels; 92% of teachers do not use travel pass for authorised trips. 5 The general impression that emerges from these observations is an obvious lack of organisation, control and accountability, all of which can compromise the performance of the system and the chances of success for the many ongoing reforms. This paper focuses on the tasks that Malagasy educators deem essential to the mission of managing for results (Government of Madagascar, 2004). The list contains seven tasks for teachers and seven for school heads (Table 2). For a teacher, these tasks include, for example, taking daily roll call, preparing the lesson of the day, monitoring student learning and helping lagging pupils. For a school head, they include keeping a register of enrolment, analysing student absences on a regular basis, following up lesson planning with teachers, reviewing pupils' performance and so on. As a strategy for keeping the analysis tractable, a 'good' teacher (or, more accurately, a minimally conscientious teacher) is defined as one who performs all seven work flow tasks that Malagasy educators consider essential to the role and likewise for a 'good' school head. In the same vein, a well-managed or a 'conscientiously managed' school is one where the school director and all of the teachers perform all of their essential tasks. complete record of their work.

According to the school survey data, only 42% of teachers perform all of the tasks deemed essential for good classroom management (Table 3). In 24% of the schools, all of the teachers perform all of their essential tasks. The share of 'conscientiously managed' schools is only 15%. Obviously, these findings reveal that many aspects of the pedagogical process are poorly managed and that far too many school personnel neglect tasks that are deemed essential for student learning. The lack of organisation, control and accountability within schools compromises the performance of the system and the chance of success for many ongoing reforms. Results reported in a companion paper indicate that the way schools are run has a significant impact on students' performance. All else remaining the same, students enrolled in a 'conscientiously managed' school score about 0.14 of a standard deviation above the sample mean, compared with their counterparts, and this management effect is much stronger for low achievers [START_REF] Lassibille | La gestion des processus pédagogiques[END_REF].

Correlates of teachers' engagement at work

The picture that emerges from the previous analysis is that teachers' engagement in teaching and teachers' effort at work are low. To examine which attributes are associated with engagement at work, the probability that a teacher will execute all of the tasks considered essential to his role is regressed on his personal characteristics and on school-level variables representing his workplace environment. 6 Specifically, the individual factors postulated to influence teacher behaviour include gender, marital status, residential location, attachment to the local community, employment conditions and professional experience, including whether the teacher had previously been employed in a private school and whether he held a second job at the time of the survey. Variables at both the classroom and school levels include the number of students in the classroom, an index of school conditions-constructed using principal components analysis based on the quality of the physical facilities of the school 7 -and the school head's management style, controlling for subdistrict-specific fixed effects. 8 These fixed effects capture unobserved characteristics that are common to all teachers within the same subdistrict and that influence teachers' engagement at work. Among these school-district effects is the management style of the subdistrict officers who supervise teaching and learning practices in schools. Under certain circumstances, teachers may self-select the schools to which they will apply and thus may have some choice in location. While the micro-level variation in teacher distribution may reflect variations in the attractiveness of specific schools, such as the availability of housing, proximity to roads and access to centres of population, it is highly unlikely that new teachers will be able to select a school on the basis of the quality of work their future peers may perform in the classroom. In the absence of self-selection on unobserved characteristics related to teachers' engagement at work, a probit model with no selection is used to adjust the probability for a teacher to execute all of the tasks considered essential to his role. Because teachers' behaviour within the same school may be correlated, and residuals may be 6 An alternative approach is to follow the methodology of [START_REF] Kling | Experimental Analysis of Neighborhood Effects[END_REF] whereby engagement at work is analysed using 'seemingly unrelated regression' for each task-either separately or in clusters of tasks. This approach was considered for this article, but the judgement of Malagasy educators that the individual tasks are an integrated package of closely connected actions required for managing the teaching and learning process was ultimately accepted. The robustness of the results presented here was also checked by considering the number of tasks performed by each teacher. Regression results not shown here to save place indicate that considering a continuum of conscientiousness does not alter the results discussed below. 7 The following items are included in the construction of the index: the structure is permanent, the number of classrooms is sufficient and the school is equipped with electricity, water, latrines and chairs for all pupils. The index ranges from about 126 in schools with all of these features to 75 in those with none of them. 8 If peer pressure is sufficiently strong, teachers' behaviour may also be affected by the presence of good teachers in a school. Because the data do not permit to separate peer effects from confounding effects that could cause individual behaviour and peer behaviour to correlate, estimating peer effects on the management of the pedagogical process is beyond the scope of this paper.

not independent within schools, standard errors of the coefficients may be biased unless this correlation is corrected for. Hence, possible intra-school correlation is taken into account by clustering standard errors at the school level. Two basic specifications are adjusted: first, a reduced model that only controls for teacher characteristics and, second, a full specification that includes class-and school-level variables. The results are shown in Table 4 along with the summary statistics of the variables for the data set. After excluding observations with missing values, the final sample includes 1,007 teachers in 294 schools.

The findings indicate that personal demographics explain little of the variation in the behaviour of school personnel. As shown in Table 4, taken together, the variables used in the analysis account for less than 2% of the variation in the probability for a teacher to perform all of the pedagogical and administrative tasks that are considered essential for a well-functioning system. To some extent, this result is in line with what is observed in other countries. To illustrate, [START_REF] Rutter | Facilitating Teacher Engagement[END_REF] finds very moderate relationships between the characteristics of teachers and teachers' commitment at work in the USA; [START_REF] Basikin | Vigor, dedication, and absorption: work engagement among secondary school English teachers in Indonesia[END_REF] obtains a similar result for secondary school teachers in Indonesia. 9 The estimated coefficients on school districts are jointly very significant, as revealed by the P-values at the bottom of the table.

Focusing now on the characteristics of individual teachers, the results from the regressions presented in Table 4 show no significant gender effect on the probability of performing the essential tasks. Several studies have argued that motivation and incentives to help the community are probably higher among local teachers because of their ties to the local area and greater sense of accountability (see, for example, [START_REF] Alcázar | Why Are Teachers Absent? Probing Service Delivery in Peruvian Primary Schools[END_REF]. However, the results of this study indicate that attachment to the local community-measured by a dummy indicating whether the teacher was born in the region where the school is located-has no significant impact on teachers' behaviour at work. 10 In a similar vein, teachers who were previously teaching in the private sector are not more likely to perform the essential tasks than their counterparts. One possible reason may be that the public sector is not able to attract the best private sector teachers or that private sector teachers who move to the public sector tend to adapt their level of work effort to the level of commitment of their new peers. Attachment to the teaching force, measured by the years of service as a teacher over the teachers' entire career and in the present school, has no significant impact on the probability of performing all of the tasks deemed essential for good classroom management. Otherwise stated, the ability of teachers to manage the pedagogical process in their classroom does not improve with accumulated experience. This result also suggests that the probability for an experienced teacher to be a good mentor is probably low and that the probability for a novice teacher to learn good practices from his senior peers in the school is probably small. Because good managerial skills with regard to the pedagogical process are not acquired through learning-by-doing and probably not through learning from others either, building skills for good classroom management would require specific actions and interventions. As teachers in Madagascar do not receive specific training on the management of the pedagogical process, ongoing pre-and in-service training programmes for teachers could include short modules on classroom management, and operational tools designed to help teachers to accomplish their tasks should be developed. Another noteworthy feature in the results presented in Table 4 is that the type of employment contract held by teachers has a significant impact on teachers' work behaviour. Based on their job status, there are two broad categories of teachers in Madagascar. The first group of teachers consists of civil service teachers who are recruited and paid by the government. The second group consists of FRAM teachers. 11 In past years, FRAM teachers were hired by the communities to respond to the lack of publicly paid teachers in the country; they were paid entirely by the parents' association through fees paid by pupils' families. Since 2002 -03, the government has contributed to the pay of FRAM teachers. Today, these teachers make up 50% of the teaching staff at the primary level, compared with only 5% in 1998. Substantial differences do exist between the two categories of teachers that are likely to affect their effectiveness. Contract teachers in Madagascar are generally less experienced than civil service teachers, with about 18 years less of professional experience than the former, on average. They have very often more years of formal schooling than civil service teachers; for example 22% of FRAM teachers have an upper secondary education diploma compared with only 9% of civil service teachers. Exposure to pre-service teacher training is relatively limited among contract teachers, with about 14% of them holding a pedagogical diploma compared with more than 85% of regular teachers. They are paid considerably less than their counterparts for doing the same job. Their monthly salary is 60% below that of civil service teachers on average, and they are paid for only 10 months out of 12.12 According to the results in Table 4, FRAM teachers are significantly less likely to execute conscientiously the tasks that are considered essential to their role. The estimates indicate that contract teachers have a probability of performing the seven essential tasks 10 percentage points lower than their counterparts, ceteris paribus. This is an important result because Madagascar, like many others developing countries, has recently hired a large number of contract teachers. The lower engagement at work of con-tract teachers may reflect a weaker attachment to the post. To a consider-able extent, it reflects lower motivation due to a combination of weak incentives, fairly limited promotion opportunities within the education sector and low job satisfaction. Results not reported here to save place indicate that: (a) the probability for a contract teacher to be integrated into the regular teaching force-and consequently to improve his economic situation-is only 13% on average; and (b) 34% of civil service teachers are highly satisfied with their job compared with 25% of contract teachers, where highly satisfied is defined as reports of job satisfaction at the level of 7 or higher on a 10-point scale.

When teacher pay is low, the incentives to generate additional income from secondary activity are generally high. In the sample, about 8% of teachers hold a second job. As expected, having a second job directly affects work effort in the main job. As shown in Table 4, teachers with a second job have a probability of executing the essential tasks 16 percentage points lower than their counterparts.

Regarding school-and class-level variables included in the model, no significant relationship is found between the number of students taught in the classroom and the way teachers handle the essential aspects of teaching. The intuition of this result is clear with regard to the nature of many of the elemental tasks teachers have to perform. Obviously, preparing lessons, discussing student learning issues with the director or monitoring student learning is to a large extent 'fixed-cost tasks'-that is, the cost of performing one of these particular tasks stays the same regardless of the number of students in the classroom. Similarly, no significant relationship is found between the overall working conditions that teachers encounter at the school-measured through an infrastructure index-and their probability of performing all of the tasks deemed essential for good classroom management. This is a somewhat unexpected result because workers are generally expected to do a better job and to be more motivated in a good physical working environment. A quadratic term of the infrastructure index was included in the model to check for possible threshold effects and non-linearities in the relationship between teachers' working conditions and engagement at work. However, results not reported here indicate no significant non-linearities in the relationship. Finally, results in Table 4 show that the principal's management style has a significant and positive impact on teachers' commitment at work. Teachers in schools administered by a 'good' school head have a probability of performing all of the essential tasks about 8 percentage points higher than their counterparts. This finding is in line with the commonly held belief that school leaders are vitally important in influencing teachers' behaviour and have a central role in developing effective schools (see, for example, [START_REF] Purkey | Effective Schools: A Review[END_REF][START_REF] Bennell | Teacher Motivation and Incentives in Sub-Saharan Africa and Asia[END_REF][START_REF] Dinham | Principal leadership for outstanding schooling outcomes in junior secondary education[END_REF]. Where teachers' motivation and incentives are low and professional qualifications are scarce, making explicit to teachers their responsibilities, giving them proper support and supervising their work are crucial elements in promoting excellence among staff. For these reasons, particular attention should be paid to the recruitment and training of head teachers in order to ensure that every school is placed under the control of a strong and engaged principal.

Conclusion

Schools face complex problems, and for the most part the solutions have not yet been found. A detailed analysis of how education is delivered in schools reveals that many aspects of the pedagogical process are poorly managed and that far too many school personnel and administrators neglect tasks deemed essential for student learning. As mentioned earlier, pupil absenteeism is poorly monitored by teachers and poorly supervised by school directors. Essential pedagogical tasks are often neglected: 20% of teachers do not prepare daily lesson plans, and school directors rarely discuss with teachers their daily lesson plans. Students' academic progress is poorly monitored, and communication from teachers to parents on student learning is often perfunctory. School directors rarely follow up with their teaching staff on student performance; more than 80% of them fail to report teacher absences to administrators at the subdistrict and district levels. In only 15% of the sample schools do all the teachers and school directors consistently perform the package of seven tasks considered essential by Malagasy educators.

The regression results clearly show that contract teachers are significantly less likely than civil service teachers to execute conscientiously the tasks that are considered essential to their role. The lower engagement at work of contract teachers may reflect a lower attachment to the post due to a combination of weak incentives, fairly limited opportunities for promotion and low job satisfaction.

It is nonetheless important to emphasise that even for civil service teachers, there is no room for complacency. The share of 'good teachers' among this group is about 45%. Moreover, the results show that the ability of teachers to manage the pedagogical process in their classroom does not improve with accumulated experience. Otherwise stated, the probability for a novice teacher to learn good practices from his senior peers in the school is probably small. The findings from this study have also shown that the principal is the key agent in the school and that leadership is vitally important in developing effective schools and in facilitating quality teaching and learning. In this regard, teachers in schools administered by a 'good' school head have a probability of performing all of the essential tasks about 8 percentage points higher than their counterparts.

There are clear signs of weak management. Various interventions could be fashioned with the goal of rationalising and tightening the work processes of teachers and making schools more effective. Improving the management of the pedagogical process would require defining and prioritising the responsibilities of teachers and school directors, with mechanisms for support and performance monitoring. In this regard, school personnel should be equipped and trained with management tools specific to their role and designed to aid in task execution and to reinforce reporting relationships among school personnel. The toolkit of operational instruments should focus on teachers' core responsibilities-for example, pedagogy, student learning and follow-up, management of instructional time, administration, school statistics and partnership with the local community-and should be designed to modify the current behaviour of personnel. The management action plan should include features that encourage the school director to pay closer attention to the work of the personnel he or she supervises, to review information supplied by a teacher and to suggest specific follow-up actions when needed. Special attention should be paid to the recruitment and training of school directors in order to ensure that every school is placed under the control of a strong and engaged principal. By increasing information flows between personnel and improving accountability, this set of interventions should lead to improvement in their behaviour. These positive behavioural changes would lead to better-functioning schools, which would positively influence the quality of education and translate into improved student learning.

Funding

Financial support from the Government of Andalucía (grants P09SEJ-4859 and SEJ-0157) is gratefully acknowledged.

Teachers' Engagement at Work in a Developing Country

The results presented here pertain to schools that did not receive any of the interventions associated with the experiment.

This information is available for up to five teachers who were randomly selected in each school.

Detailed data on artefacts, such as those collected in the framework of this research, are extremely rare. This rarity is not surprising given that teachers may be unwilling to part with a source of information that provides a relatively

For more information on teachers' absence in Madagascar, see, for example, World Bank (2008).

In Madagascar, teachers are routinely granted permission for travel to collect their salaries at designated localities. In the absence of supervision of such travel, the trips may result in excessive absences from the classroom.

However, the definition of engagement at work used by these authors departs from the one used here, as they also take into account attitudes and predispositions at work

A similar result is obtained when measuring attachment to the local community by a dummy indicating whether the teacher was born in the community where the school is located.

FRAM is the acronym for Fikambanan'ny Ray Amandrenin'ny Mpianatra, the schoolbased parent-teacher association.

For more details on FRAM teachers, see Government ofMadagascar (2008) and World Bank (2002).Teachers' Engagement at Work in a Developing Country

Acknowledgements

The author is grateful to the World Bank and the Ministry of Education of Madagascar for providing access to the AGEMAD (Amélioration de la Gestion de l'Education à Madagascar) database used here. The author thanks Cornelia Jesse, Lucia Navarro Gómez, Jee-Peng Tan and participants at the Fourth Workshop on Improving Education Management in African Countries for helpful discussions and comments.