The Discovery of Gut Microbial Metabolites as Modulators of Host Susceptibility to Acetaminophen-Induced Hepatotoxicity.

The mammalian gut microbiota plays diverse and essential roles in modulating host physiology. Key mediators determining the outcome of the microbiota-host interactions are the small molecule metabolites produced by the gut microbiota. The liver is a major organ exposed to gut microbial metabolites, and it serves as the nexus for maintaining healthy interactions between the gut microbiota and the host. At the same time, the liver is the primary target of potentially harmful gut microbial metabolites. In this review, we provide an up-to-date list of gut microbial metabolites that have been identified to either increase or decrease host susceptibility to acetaminophen (APAP)-induced liver injury. The signaling pathways and molecular factors involved in the progression of APAP-induced hepatotoxicity are well-established, and we propose that the mouse model of APAP-induced hepatotoxicity serves as a model system for uncovering gut microbial metabolites with previously unknown functions. Furthermore, we envision that gut microbial metabolites identified to alter APAP-induced hepatotoxicity likely have broader implications in other liver diseases. SIGNIFICANCE STATEMENT: This review provides an overview of the role of the gut microbiota in modulating the host susceptibility to acetaminophen (APAP)-induced liver injury. It focuses on the roles of gut bacterial small molecule metabolites as mediators of the interaction between the gut microbiota and the liver. It also illustrates the utility of APAP-induced liver injury as a model to identify gut microbial metabolites with biological function.

Retinoic Acid Differentially Regulates the Migration of Innate Lymphoid Cell Subsets to the Gut.

Distinct groups of innate lymphoid cells (ILCs) such as ILC1, ILC2, and ILC3 populate the intestine, but how these ILCs develop tissue tropism for this organ is unclear. We report that prior to migration to the intestine ILCs first undergo a "switch" in their expression of homing receptors from lymphoid to gut homing receptors. This process is regulated by mucosal dendritic cells and the gut-specific tissue factor retinoic acid (RA). This change in homing receptors is required for long-term population and effector function of ILCs in the intestine. Only ILC1 and ILC3, but not ILC2, undergo the RA-dependent homing receptor switch in gut-associated lymphoid tissues. In contrast, ILC2 acquire gut homing receptors in a largely RA-independent manner during their development in the bone marrow and can migrate directly to the intestine. Thus, distinct programs regulate the migration of ILC subsets to the intestine for regulation of innate immunity.

Cutting Edge: The Expression of Transcription Inhibitor GFI1 Is Induced by Retinoic Acid to Rein in Th9 Polarization.

IL-9, produced mainly by specialized T cells, mast cells, and group 2 innate lymphoid cells, regulates immune responses, including anti-helminth and allergic responses. Polarization of naive CD4 T cells into IL-9-producing T cells (Th9s) is induced by IL-4 and TGF-ß1 or IL-1ß. In this article, we report that the transcription factor growth factor-independent 1 transcriptional repressor (GFI1) plays a negative role in mouse Th9 polarization. Moreover, the expression of GFI1 is controlled by liganded RARα, allowing GFI1 to mediate the negative effect of retinoic acid on IL-9 expression. The Gfi1 gene has multiple RARα binding sites in the promoter region for recruiting nuclear coactivator steroid receptor coactivator-3 and p300 for histone epigenetic modifications in a retinoic acid-dependent manner. Retinoic acid-induced GFI1 binds the Il9 gene and suppresses its expression. Thus, GFI1 is a novel negative regulator of Il9 gene expression. The negative GFI1 pathway for IL-9 regulation provides a potential control point for Th9 activity.

Digital Health in Diabetes and Cardiovascular Disease.

BACKGROUND: Digital health technologies are rapidly evolving and transforming the care of diabetes and cardiovascular disease (CVD). PURPOSE OF THE REVIEW: In this review, we discuss emerging approaches incorporating digital health technologies to improve patient outcomes through a more continuous, accessible, proactive, and patient-centered approach. We discuss various mechanisms of potential benefit ranging from early detection to enhanced physiologic monitoring over time to helping shape important management decisions and engaging patients in their care. Furthermore, we discuss the potential for better individualization of management, which is particularly important in diseases with heterogeneous and complex manifestations, such as diabetes and cardiovascular disease. This narrative review explores ways to leverage digital health technology to better extend the reach of clinicians beyond the physical hospital and clinic spaces to address disparities in the diagnosis, treatment, and prevention of diabetes and cardiovascular disease. CONCLUSION: We are at the early stages of the shift to digital medicine, which holds substantial promise not only to improve patient outcomes but also to lower the costs of care. The review concludes by recognizing the challenges and limitations that need to be addressed for optimal implementation and impact. We present recommendations on how to navigate these challenges as well as goals and opportunities in utilizing digital health technology in the management of diabetes and prevention of adverse cardiovascular outcomes.

The Butyrate-Producing Bacterium Clostridium butyricum Suppresses Clostridioides difficile Infection via Neutrophil- and Antimicrobial Cytokine-Dependent but GPR43/109a-Independent Mechanisms.

Short-chain fatty acids, such as butyrate, are major gut microbial metabolites that are beneficial for gastrointestinal health. Clostridium butyricum MIYAIRI588 (CBM588) is a bacterium that produces a robust amount of butyrate and therefore has been used as a live biotherapeutic probiotic in clinical settings. Clostridioides difficile causes life-threatening diarrhea and colitis. The gut resident microbiota plays a critical role in the prevention of C. difficile infection (CDI), as the disruption of the healthy microbiota by antibiotics greatly increases the risk for CDI. We report that CBM588 treatment in mice significantly improved clinical symptoms associated with CDI and increased the number of neutrophils and Th1 and Th17 cells in the colonic lamina propria in the early phase of CDI. The protective effect of CBM588 was abolished when neutrophils, IFN-γ, or IL-17A were depleted, suggesting that induction of the immune reactants is required to elicit the protective effect of the probiotic. The administration of tributyrin, which elevates the concentration of butyrate in the colon, also increased the number of neutrophils in the colonic lamina propria, indicating that butyrate is a potent booster of neutrophil activity during infection. However, GPR43 and GPR109a, two G protein-coupled receptors activated by butyrate, were dispensable for the protective effect of CBM588. These results indicate that CBM588 and butyrate suppress CDI, in part by boosting antimicrobial innate and cytokine-mediated immunity.

Differential Risk of Cancer Associated with Glucagon-like Peptide-1 Receptor Agonists: Analysis of Real-world Databases.

BACKGROUND: Glucagon-like peptide 1 receptor agonists (GLP1Ra) are commonly used in type 2 diabetes mellitus (T2DM). However, differential risk of various cancers among GLP1Ra recipients is unknown. METHODS: We inquired an aggregated electronic health record database, Explorys, and compared the adjusted odds ratio (aOR) of cancers between GLP1Ra and metformin users. Findings were validated in the FDA Adverse Event Reporting System (FDA FAERS). RESULTS: From 1/2005 to 6/2019, we identified 619 340 and 64 230 patients in the metformin and GLP1Ra group, respectively. Within 5 years of starting antidiabetic medications, GLP1Ra was associated with significantly lower incident risk of prostate (aOR 0.81, p = .03), lung (aOR 0.81, p = .05), and colon cancer (aOR 0.85, p = .03), while the risk of thyroid cancer was significantly higher (aOR 1.65, p < .01). Similar findings were seen in the FDA FAERS database, where GLP1Ra was associated with lower risk of prostate (aOR 0.72, p = .08), lung (aOR 0.52, p < .01), colon cancer (aOR 0.82, p = .31), and higher risk of thyroid cancer (aOR 4.33, p < .01). In addition, with longer duration of GLP1Ra use, the risk of prostate, lung, and colon cancer further decreased, suggesting an exposure duration-response relationship. CONCLUSIONS: GLP1Ra is associated with lower risks of prostate, lung, and colon cancer, but higher risk of thyroid cancer.

IL-4-BATF signaling directly modulates IL-9 producing mucosal mast cell (MMC9) function in experimental food allergy.

BACKGROUND: This study group has previously identified IL-9-producing mucosal mast cell (MMC9) as the primary source of IL-9 to drive intestinal mastocytosis and experimental IgE-mediated food allergy. However, the molecular mechanisms that regulate the expansion of MMC9s remain unknown. OBJECTIVES: This study hypothesized that IL-4 regulates MMC9 development and MMC9-dependent experimental IgE-mediated food allergy. METHODS: An epicutaneous sensitization model was used and bone marrow reconstitution experiments were performed to test the requirement of IL-4 receptor α (IL-4Rα) signaling on MMC9s in experimental IgE-mediated food allergy. Flow cytometric, bulk, and single-cell RNA-sequencing analyses on small intestine (SI) MMC9s were performed to illuminate MMC9 transcriptional signature and the effect of IL-4Rα signaling on MMC9 function. A bone marrow-derived MMC9 culture system was used to define IL-4-BATF signaling in MMC9 development. RESULTS: Epicutaneous sensitization- and bone marrow reconstitution-based models of IgE-mediated food allergy revealed an IL-4 signaling-dependent cell-intrinsic effect on SI MMC9 accumulation and food allergy severity. RNA-sequencing analysis of SI-MMC9s identified 410 gene transcripts reciprocally regulated by IL-4 signaling, including Il9 and Batf. Insilico analyses identified a 3491-gene MMC9 transcriptional signature and identified 2 transcriptionally distinct SI MMC9 populations enriched for metabolic or inflammatory programs. Employing an in vitro MMC9-culture model system showed that generation of MMC9-like cells was induced by IL-4 and this was in part dependent on BATF. CONCLUSIONS: IL-4Rα signaling directly modulates MMC9 function and exacerbation of experimental IgE-mediated food allergic reactions. IL-4Rα regulation of MMC9s is in part BATF-dependent and occurs via modulation of metabolic transcriptional programs.

Control of Tissue-Resident Invariant NKT Cells by Vitamin A Metabolites and P2X7-Mediated Cell Death.

Invariant NKT (iNKT) cells provide rapid innate T cell responses to glycolipid Ags from host cells and microbes. The numbers of CD1d-restricted iNKT cells are tightly controlled in mucosal tissues, but the mechanisms have been largely unclear. We found that vitamin A is a dominant factor that controls the population size of mucosal iNKT cells in mice. This negative regulation is mediated by the induction of the purinergic receptor P2X7 on iNKT cells. The expression of P2X7 is particularly high on intestinal iNKT cells, making iNKT cells highly susceptible to P2X7-mediated cell death. In vitamin A deficiency, iNKT cells fail to express P2X7 and are, therefore, resistant to P2X7-mediated cell death, leading to iNKT cell overpopulation. This phenomenon is most prominent in the intestine. We found that iNKT cells are divided into CD69+ sphingosine-1-phosphate receptor 1 (S1P1)- tissue resident and CD69- S1P1+ nonresident iNKT cells. The CD69+ S1P1- tissue-resident iNKT cells highly express P2X7 and are effectively controlled by the P2X7 pathway. The regulation of iNKT cells by vitamin A by the P2X7 pathway is important to prevent aberrant expansion of effector cytokine-producing iNKT cells. Our findings identify a novel role of vitamin A in regulating iNKT cell homeostasis in many tissues throughout the body.

Weak Microbial Metabolites: a Treasure Trove for Using Biomimicry to Discover and Optimize Drugs.

For decades, traditional drug discovery has used natural product and synthetic chemistry approaches to generate libraries of compounds, with some ending as promising drug candidates. A complementary approach has been to adopt the concept of biomimicry of natural products and metabolites so as to improve multiple drug-like features of the parent molecule. In this effort, promiscuous and weak interactions between ligands and receptors are often ignored in a drug discovery process. In this Emerging Concepts article, we highlight microbial metabolite mimicry, whereby parent metabolites have weak interactions with their receptors that then have led to discrete examples of more potent and effective drug-like molecules. We show specific examples of parent-metabolite mimics with potent effects in vitro and in vivo. Furthermore, we show examples of emerging microbial ligand-receptor interactions and provide a context in which these ligands could be improved as potential drugs. A balanced conceptual advance is provided in which we also acknowledge potential pitfalls-hyperstimulation of finely balanced receptor-ligand interactions could also be detrimental. However, with balance, we provide examples of where this emerging concept needs to be tested. SIGNIFICANCE STATEMENT: Microbial metabolite mimicry is a novel way to expand on the chemical repertoire of future drugs. The emerging concept is now explained using specific examples of the discovery of therapeutic leads from microbial metabolites.

Microbial metabolites, short-chain fatty acids, restrain tissue bacterial load, chronic inflammation, and associated cancer in the colon of mice.

The intestinal immune system is regulated by microbes and their metabolites. The roles of gut microbial metabolites in regulating intestinal inflammation and tumorigenesis are incompletely understood. We systematically studied the roles of short-chain fatty acids (SCFAs) and their receptors (GPR43 or GPR41) in regulating tissue bacterial load, acute versus chronic inflammatory responses, and intestinal cancer development. SCFA receptor-, particularly GPR43-, deficient mice were defective in mounting appropriate acute immune responses to promote barrier immunity, and developed uncontrolled chronic inflammatory responses following epithelial damage. Further, intestinal carcinogenesis was increased in GPR43-deficient mice. Dietary fiber and SCFA administration suppressed intestinal inflammation and cancer in both GPR43-dependent and independent manners. The beneficial effect of GPR43 was not mediated by altered microbiota but by host tissue cells and hematopoietic cells to a lesser degree. We found that inability to suppress commensal bacterial invasion into the colonic tissue is associated with the increased chronic Th17-driven inflammation and carcinogenesis in the intestine of GPR43-deficient mice. In sum, our results reveal the beneficial function of the SCFA-GPR43 axis in suppressing bacterial invasion and associated chronic inflammation and carcinogenesis in the colon.

Migration and Tissue Tropism of Innate Lymphoid Cells.

Innate lymphoid cell (ILCs) subsets differentially populate various barrier and non-barrier tissues, where they play important roles in tissue homeostasis and tissue-specific responses to pathogen attack. Recent findings have provided insight into the molecular mechanisms that guide ILC migration into peripheral tissues, revealing common features among different ILC subsets as well as important distinctions. Recent studies have also highlighted the impact of tissue-specific cues on ILC migration, and the importance of the local immunological milieu. We review these findings here and discuss how the migratory patterns and tissue tropism of different ILC subsets relate to the development and differentiation of these cells, and to ILC-mediated tissue-specific regulation of innate and adaptive immune responses. In this context we outline open questions and important areas of future research.

Immune regulation by microbiome metabolites.

Commensal microbes and the host immune system have been co-evolved for mutual regulation. Microbes regulate the host immune system, in part, by producing metabolites. A mounting body of evidence indicates that diverse microbial metabolites profoundly regulate the immune system via host receptors and other target molecules. Immune cells express metabolite-specific receptors such as P2X7 , GPR41, GPR43, GPR109A, aryl hydrocarbon receptor precursor (AhR), pregnane X receptor (PXR), farnesoid X receptor (FXR), TGR5 and other molecular targets. Microbial metabolites and their receptors form an extensive array of signals to respond to changes in nutrition, health and immunological status. As a consequence, microbial metabolite signals contribute to nutrient harvest from diet, and regulate host metabolism and the immune system. Importantly, microbial metabolites bidirectionally function to promote both tolerance and immunity to effectively fight infection without developing inflammatory diseases. In pathogenic conditions, adverse effects of microbial metabolites have been observed as well. Key immune-regulatory functions of the metabolites, generated from carbohydrates, proteins and bile acids, are reviewed in this article.

Chronically Elevated Levels of Short-Chain Fatty Acids Induce T Cell-Mediated Ureteritis and Hydronephrosis.

Short-chain fatty acids (SCFAs) are major products of gut microbial fermentation and profoundly affect host health and disease. SCFAs generate IL-10(+) regulatory T cells, which may promote immune tolerance. However, SCFAs can also induce Th1 and Th17 cells upon immunological challenges and, therefore, also have the potential to induce inflammatory responses. Because of the seemingly paradoxical SCFA activities in regulating T cells, we investigated, in depth, the impact of elevated SCFA levels on T cells and tissue inflammation in mice. Orally administered SCFAs induced effector (Th1 and Th17) and regulatory T cells in ureter and kidney tissues, and they induced T cell-mediated ureteritis, leading to kidney hydronephrosis (hereafter called acetate-induced renal disease, or C2RD). Kidney hydronephrosis in C2RD was caused by ureteral obstruction, which was, in turn, induced by SCFA-induced inflammation in the ureteropelvic junction and proximal ureter. Oral administration of all major SCFAs, such as acetate, propionate, and butyrate, induced the disease. We found that C2RD development is dependent on mammalian target of rapamycin activation, T cell-derived inflammatory cytokines such as IFN-γ and IL-17, and gut microbiota. Young or male animals were more susceptible than old or female animals, respectively. However, SCFA receptor (GPR41 or GPR43) deficiency did not affect C2RD development. Thus, SCFAs, when systemically administered at levels higher than physiological levels, cause dysregulated T cell responses and tissue inflammation in the renal system. The results provide insights into the immunological and pathological effects of chronically elevated SCFAs.

Prognostic Significance of Hemodynamic and Clinical Stages in the Prediction of Hepatocellular Carcinoma.

BACKGROUND & GOALS: Early identification of hepatocellular carcinoma (HCC) is associated with improved survival for patients with chronic liver disease (CLD). We evaluated the prognostic significance of hemodynamic stage (HS) and clinical stage (CS) in predicting HCC in CLD patients. METHODS: Between January 2006 and May 2014, 801 patients with CLD who underwent hepatic venous pressure gradient (HVPG) measurement were prospectively enrolled. HS was classified by HVPG (mm Hg) as follows: HS-1 (HVPG≤6), HS-2 (612 mm Hg (P=0.033, OR=2.17), CS>2 (P=0.039, OR=2.36), and alpha-fetoprotein (AFP; P=0.017, OR=1.01) were significant predictors of HCC development in all patients. For patients with cirrhosis, ascites aggravation (OR=2.51), HVPG >12 mm Hg (OR=2.46), and CS >2 (OR=2.62) were correlated with HCC development. Areas under receiver operating characteristic curves of the prediction-model, CS, HVPG score, and AFP were 0.797, 0.707, 0.701, and 0.653, respectively. CONCLUSIONS: HCC development correlates with advancing liver fibrosis or disease as measured by HS and CS. In addition, ascites aggravation and elevated AFP appears to be associated with increased incidence of HCC.

Cutting edge: progesterone directly upregulates vitamin d receptor gene expression for efficient regulation of T cells by calcitriol.

The two nuclear hormone receptor ligands progesterone and vitamin D (vit.D) play important roles in regulating T cells. The mechanism that connects these two hormones in regulating T cells has not been established. In this study, we report that progesterone is a novel inducer of vit.D receptor (VDR) in T cells and makes T cells highly sensitive to calcitriol. At the molecular level, the induction by progesterone is mediated by two progesterone receptor-binding elements in the intron region after the first noncoding exon of the human VDR gene. Increased expression of VDR by progesterone allows highly sensitive regulation of T cells by vit.D even when vit.D levels are suboptimal. This novel regulatory pathway allows enhanced induction of regulatory T cells but suppression of Th1 and Th17 cells by the two nuclear hormones. The results have significant ramifications in effective regulation of T cells to prevent adverse immune responses during pregnancy.

Authentic GITR Signaling Fails To Induce Tumor Regression unless Foxp3+ Regulatory T Cells Are Depleted.

The glucocorticoid-induced TNFR family-related protein (GITR, TNFRSF18, CD357) is expressed on effector and regulatory T (Treg) cells. Previous studies demonstrated that GITR triggering by anti-GITR mAb enhanced T and B cell-mediated immune responses. GITR-deficient T cells, however, also proliferate more than normal T cells, and this effect is unexplained. Because the activities of mAbs are controlled by their Fc regions, the true effect of GITR signaling needs to be determined by examining its interaction with authentic ligand. Therefore, we generated a pentamerized form of the GITRL extracellular domain (pGITRL) for ligation to GITR and compared its effect on T cells with that of anti-GITR mAb. The pGITRL was more effective than anti-GITR mAb in enhancing the proliferation of effector and regulatory cells in vitro and in vivo. Nonetheless, the growth of MC38 adenocarcinoma cells in vivo was only suppressed for initial 15 d by pGITRL, whereas it was suppressed indefinitely by anti-GITR mAb. Detailed analysis revealed that pGITRL induced extensive proliferation of Foxp3(+)CD4(+) Treg cells and led to the accumulation of activated Treg cells in tumor tissue and draining lymph nodes. Because GITR signaling could not neutralize the suppressive activity of activated Treg cells, pGITRL seems to lose its adjuvant effect when sufficient activated Treg cells have accumulated in the lymph nodes and tumor tissue. Indeed, the antitumor effects of pGITRL were markedly enhanced by depleting CD4(+) cells. These results suggest that GITR signaling has stimulatory effects on effector T cells and inhibitory effects through Treg cells.

Short-chain fatty acids activate GPR41 and GPR43 on intestinal epithelial cells to promote inflammatory responses in mice.

BACKGROUND & AIMS: Short-chain fatty acids (SCFAs), the most abundant microbial metabolites in the intestine, activate cells via G-protein-coupled receptors (GPRs), such as GPR41 and GPR43. We studied regulation of the immune response by SCFAs and their receptors in the intestines of mice. METHODS: Inflammatory responses were induced in GPR41(-/-), GPR43(-/-), and C57BL6 (control) mice by administration of ethanol; 2, 4, 6-trinitrobenzene sulfonic-acid (TNBS); or infection with Citrobacter rodentium. We examined the effects of C rodentium infection on control mice fed SCFAs and/or given injections of antibodies that delay the immune response. We also studied the kinetics of cytokine and chemokine production, leukocyte recruitment, intestinal permeability, and T-cell responses. Primary colon epithelial cells were isolated from GPR41(-/-), GPR43(-/-), and control mice; signaling pathways regulated by SCFAs were identified using immunohistochemical, enzyme-linked immunosorbent assay, and flow cytometry analyses. RESULTS: GPR41(-/-) and GPR43(-/-) mice had reduced inflammatory responses after administration of ethanol or TNBS compared with control mice, and had a slower immune response against C rodentium infection, clearing the bacteria more slowly. SCFAs activated intestinal epithelial cells to produce chemokines and cytokines in culture and mice after administration of ethanol, TNBS, or C rodentium. These processes required GPR41 and GPR43 and were required to recruit leukocytes and activate effector T cells in the intestine. GPR41 and GPR43 activated extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase signaling pathways in epithelial cells to induce production of chemokines and cytokines during immune responses. CONCLUSIONS: SCFAs activate GPR41 and GPR43 on intestinal epithelial cells, leading to mitogen-activated protein kinase signaling and rapid production of chemokines and cytokines. These pathways mediate protective immunity and tissue inflammation in mice.

Retinoic acid promotes the development of Arg1-expressing dendritic cells for the regulation of T-cell differentiation.

Arginase I (Arg1), an enzyme expressed by many cell types including myeloid cells, can regulate immune responses. Expression of Arg1 in myeloid cells is regulated by a number of cytokines and tissue factors that influence cell development and activation. Retinoic acid, produced from vitamin A, regulates the homing and differentiation of lymphocytes and plays important roles in the regulation of immunity and immune tolerance. We report here that optimal expression of Arg1 in DCs requires retinoic acid. Induction of Arg1 by retinoic acid is directly mediated by retinoic acid-responsive elements in the 5' noncoding region of the Arg1 gene. Arg1, produced by DCs in response to retinoic acid, promotes the generation of FoxP3(+) regulatory T (Treg) cells. Importantly, blocking the retinoic acid receptor makes DCs hypo-responsive to known inducers of Arg1 such as IL-4 and GM-CSF in Arg1 expression. We found that intestinal CD103(+) DCs that are known to produce retinoic acid highly express Arg1. Our results establish retinoic acid as a key signal in expression of Arg1 in DCs.

Circadian-clock-controlled endocrine and cytokine signals regulate multipotential innate lymphoid cell progenitors in the bone marrow.

Innate lymphoid cells (ILCs), strategically positioned throughout the body, undergo population declines over time. A solution to counteract this problem is timely mobilization of multipotential progenitors from the bone marrow. It remains unknown what triggers the mobilization of bone marrow ILC progenitors (ILCPs). We report that ILCPs are regulated by the circadian clock to emigrate and generate mature ILCs in the periphery. We found that circadian-clock-defective ILCPs fail to normally emigrate and generate ILCs. We identified circadian-clock-controlled endocrine and cytokine cues that, respectively, regulate the retention and emigration of ILCPs at distinct times of each day. Activation of the stress-hormone-sensing glucocorticoid receptor upregulates CXCR4 on ILCPs for their retention in the bone marrow, while the interleukin-18 (IL-18) and RORα signals upregulate S1PR1 on ILCPs for their mobilization to the periphery. Our findings establish important roles of circadian signals for the homeostatic efflux of bone marrow ILCPs.