Neutrophil homeostasis represents a delicately regulated balance among a series of processes, including granulopoiesis in the BM, neutrophil release into blood, neutrophil migration from the blood into tissues, and neutrophil apoptosis and clearance under both normal and inflammatory states. Tight control of neutrophils under normal and inflammatory conditions is a critical component of innate immunity. Hence, failure of neutrophil regulation may contribute to inflammatory diseases or worsening of infection. In contrast to the granulopoiesis that occurs in response to severe inflammatory stimuli, steady-state homeostasis employs mechanisms by which neutrophils are mobilized from BM to blood to maintain effective neutrophil number (which may reflect neutrophils in tissues as well as those in the circulation) despite routine stresses. Furthermore, neutrophil homeostasis may also include mechanisms by which neutrophil numbers rapidly decline upon resolution of inflammation in tissues and circulation (3
Recent advances in identifying the feedback control mechanisms regulating neutrophil production and release have indicated an important role for the IL-17/G-CSF pathway. In this study we uncover another layer in the regulation of neutrophil homeostasis via a feedback loop among Cxcr2, Cxcl5, and the IL-17/G-CSF axis. Specifically, we have demonstrated the critical role of neutrophil Cxcr2 and nonhematopoietic resident cell–derived Cxcl5 in regulating the IL-17/G-CSF axis and neutrophil homeostasis under basal conditions and, in turn, the essential role of this axis in Cxcl5-regulated neutrophil homeostasis. We propose a model in which Cxcr2, its ligands, and commensal bacteria control neutrophil homeostasis through the IL-17/G-CSF axis, primarily at mucosal sites (Figure ). Based on our findings, we propose that at mucosal sites such as gut and lung, resident cells secrete Cxcr2 ligands to attract neutrophils into tissues to inhibit the IL-17/G-CSF axis, thus regulating neutrophil homeostasis under normal conditions. In this feedback loop, enterocyte-derived Cxcl5 in the gut, but not AE II cell–derived Cxcl5 in the lung, is required for inhibition of IL-17 production, while other Cxcr2 ligands may be responsible for Cxcr2-mediated regulation of IL-17/G-CSF axis in the lung. In addition, commensal bacteria are required for the activation of IL-17/G-CSF axis caused by Cxcr2 deficiency, and IL-1 and IL-23 signaling may serve to induce IL-17 production in vivo.
The proposed roles of CXCR2, CXCL5, and commensal bacteria in neutrophil homeostasis under normal conditions.
Granulopoiesis in the BM is an essential first step in neutrophil homeostasis. While we found extremely large numbers and percentages of neutrophils in the BM of Cxcr2–/–
mice, this represents the net result of both enhanced granulopoiesis, as we show here, and release into the circulation. Link and colleagues showed that neutrophil Cxcr2 is required for G-CSF–mediated neutrophil mobilization from the BM to the blood (4
). Therefore the high neutrophil percentages (about 90%) in the BM of Cxcr2–/–
mice are due not only to G-CSF–induced granulopoiesis, but also to the additive effect of defective G-CSF–mediated neutrophil mobilization to the blood. The defect in release also contributes to a delay in the reduction of BM neutrophil percentages upon anti–IL-17A antibody treatment in Cxcr2–/–
mice compared with that in Cxcl5–/–
mice, despite the dramatically reduced plasma G-CSF in Cxcr2–/–
mice (Figure , B–E). Furthermore, the defective neutrophil release in Cxcr2–/–
mice also reduces the neutrophil numbers in the blood, which may explain the mild neutrophilia (a 2- to 4-fold increase relative to WT mice) observed in Cxcr2–/–
mice, which is in contrast to the severe neutrophilia (about 20-fold increase relative to WT mice) observed in Itgb2–/–
mice, despite the overwhelmingly dominant neutrophil population in the BM of Cxcr2–/–
Based on the findings of Stark et al. (13
), a critical step in neutrophil homeostasis is neutrophil migration to sites of interest. Our data suggest the critical role of neutrophil Cxcr2 and nonhematopoietic resident cell–derived Cxcr2 ligands such as Cxcl5 in this process. We propose that resident cells in the gut and lung express Cxcr2 ligands that attract neutrophils to migrate into the tissues, where they regulate the IL-17/G-CSF axis and neutrophil homeostasis. The dramatic increase in IL-17A/G-CSF and BM neutrophil percentages in the absence of Cxcr2 or Cxcl5 is the evidence of interruption of this feedback loop. In addition, we found that enterocytes in the ileum express Cxcl5 under basal conditions, consistent with a previous report that Cxcl5 was expressed in colonic enterocytes in a murine colitis model (30
), suggesting that enterocyte-derived Cxcl5 mediates neutrophil transmigration into the gut to regulate steady-state neutrophil homeostasis.
While basal granulopoiesis is not thought to be responsive to the environment, we have shown here that the commensal bacteria of the gut serve a role in the regulation of the IL-17/G-CSF axis. It has been demonstrated that commensal bacteria are important for shaping intestinal immune responses under normal and inflammatory states (31
). Segmented filamentous bacteria were recently identified to promote Th17 cell differentiation (32
); however, how commensal bacteria affect IL-17–producing cell differentiation and IL-17 production under normal and inflammatory conditions remains poorly understood. Here we showed that depletion of commensal bacteria through antibiotic treatment significantly decreased systemic IL-17/G-CSF expression, BM neutrophil percentages, and peripheral neutrophilia in Cxcr2–/–
mice (Figure ), which is consistent with previous reports that germ-free Cxcr2–/–
mice lost the neutrophilia phenotype observed in SPF Cxcr2–/–
mice. These data suggest that commensal microbiota may contribute to signaling for IL-17 production in the gut, likely through pattern recognition receptors, thus regulating neutrophil homeostasis under normal conditions. WT mice administered antibiotics also exhibited decreased plasma baseline G-CSF (Figure ). Cxcl5 deficiency led to increased IL-17–producing cells in the terminal ileum, but not in the lung, providing novel insights that different mucosal sites might utilize different Cxcr2 ligands (secreted by resident epithelial cells) to attract neutrophils into the tissues to regulate IL-17/G-CSF axis and neutrophil homeostasis under normal conditions. However, the signals used to attract neutrophils to sites where they may influence IL-23 and IL-1β expression by tissue macrophages or dendritic cells, and which tissues are involved, remain unclear and warrant further investigation.
In contrast to the role of the terminal ileum in the induction of IL-17, we observed that Cxcl5 regulation of IL-17 does not extend to the distal lung. Consistent with our prior work (27
), we demonstrate here that lung AE II cells express Cxcl5 at baseline. In Cxcl5–/–
mice, however, dysregulation of IL-17 occurred only in the gut and not in the lung. We suspect that the inability of lung-derived Cxcl5 to inhibit IL-17 production may result from the relative sterility of the alveolar space limiting the activation of IL-17A/G-CSF axis, but we cannot at this time rule out the possibility that an alveolar microbiome exists and modifies IL-17A expression. We previously demonstrated high levels of another Cxcr2 ligand, lungkine (which is constitutively expressed by bronchial epithelial cells; ref. 35
), in the BALF of normal mice (21
). In addition, 2 other Cxcr2 ligands in mice, Cxcl1 and Cxcl2, can be induced by G-CSF (4
), implying that they may be involved in neutrophil release from BM. Whether Cxcl1 or Cxcl2 plays a role in regulating the IL-17/G-CSF axis and steady-state neutrophil homeostasis will require further investigation of their respective gene-targeted mice.
IL-17A–producing cells have been advanced as important controllers of inflammatory signaling (15
). The explosion of data on the varied lineages of these cells indicates that many cells are candidates for production of IL-17A in our model. In contrast to a recent study in a different model (13
), we found that in our model, not only did Th17 and γδ T cells express IL-17A in lung, gut, and spleen, but cells that were not T cells from lung and gut were also able to generate IL-17. The identity of these cells remains unclear and is the subject of ongoing work. The signals responsible for IL-17–producing cells are somewhat better established, and IL-23 has emerged as an important mediator. In our model, we do not detect a significant increase for IL-23 expression in either Cxcr2–/–
mice, perhaps because of detection limits or posttranscriptional regulation of this potent cytokine. However, our data highlight the potential importance of both IL-1 and IL-23 signaling in stimulation of IL-17–producing cells.
Recently it has been shown that IL-1 signaling is crucial for IL-17 production and differentiation of early Th17 cells (25
), IL-17–producing γδ T cells, and invariant NKT cells upon synergizing with IL-23 (24
). In these studies, the Th17 cells or IL-17–producing γδ T cells from spleens or lymph nodes significantly increased upon stimulation of IL-1β/IL-23 for 3 days. Here we stimulated splenocytes from WT, Cxcl5–/–
, and Cxcr2–/–
mice for only 5 hours with IL-1β, IL-23, IL-1β/IL-23, and PMA/calcium (Figure ). Only Cxcr2–/–
mice had an increased percentage of IL-17A–producing cells (mostly CD3+
T cells) in response to IL-1β/IL-23 stimulation, albeit less than that upon stimulation of PMA/calcium ionophore, which suggests that combined signals may be required for optimal IL-17A expression.
In summary, we present a model in which Cxcr2, Cxcl5, and commensal bacteria regulate the IL-17/G-CSF axis and neutrophil homeostasis under basal conditions (see Figure for a proposed scheme). This model predicts that the local environment, sensed at mucosal sites, contributes to setting the signals for neutrophil generation. It would be prudent to consider that the intensity of these baseline signals may also influence, through commensal bacteria, the organismal response to severe stress such as pneumonia or sepsis. This question will be the subject of further study.