Differentiation and activation of eosinophils in the human bone marrow during experimental human endotoxemia

Acute infection is characterized by eosinopenia. However, the underlying mechanism(s) are poorly understood and it is unclear whether decreased mobilization/production of eosinophils in the bone marrow (BM) and/or increased homing to the tissues play a role. The objective of this study was to investigate the differentiation and activation status of eosinophils in the human BM and blood upon experimental human endotoxemia, a standardized, controlled, and reproducible model of acute systemic inflammation. A BM aspirate and venous blood was obtained from seven healthy volunteers before, 4 h after, and 1 week after intravenous challenge with 2 ng/kg endotoxin. Early progenitors (CD34+/IL‐5Rα+), eosinophil promyelocytes, myelocytes, metamyelocytes, and mature eosinophils were identified and quantified in the bone marrow and blood samples using flowcytometry based on specific eosinophil markers (CD193 and IL‐5Rα). Activation status was assessed using antibodies against known markers on eosinophils: Alpha‐4 (CD49d), CCR3 (CD193), CR1 (CD35), CEACAM‐8 (CD66b), CBRM 1/5 (activation epitope of MAC‐1), and by plasma cytokine analysis. Four hours after endotoxin administration, numbers of mature eosinophils in the blood and in the BM markedly declined compared with baseline, whereas numbers of all eosinophil progenitors did not change. The remaining eosinophils did not show signs of activation or degranulation despite significantly increased circulating levels of eotaxin‐1. Furthermore, the expression of CD49d and CD193 on eosinophils was lower compared to baseline, but normalized after 7 days. Together these data imply that circulatory eosinopenia after an innate immune challenge is mediated by CD49d‐mediated homing of eosinophils to the tissues.


INTRODUCTION
Eosinophils are cytotoxic granulocytes that are typically involved in host defense against multicellular parasites. 1 Apart from their immune function, eosinophils are also thought to be important for homeostatic functions, 2 such as maintenance of metabolic homeostasis, 3 tissue remodeling 4 and T-cell selection in the thymus. 5 On the other hand, eosinophils play a pathological role in diseases such as asthma and eosinophilic esophagitis. 6,7 In asthma, these cells are Abbreviations: BM, bone marrow; EoPs, eosinophil progenitors.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. c 2020 The Authors. Journal of Leukocyte Biology published by Wiley Periodicals, Inc. on behalf of Society for Leukocyte Biology recruited to the airways after allergic or nonallergic stimuli and are responsible for bronchial airway inflammation. 8 In eosinophilic esophagitis, eosinophils are present in the esophageal tissue but their role in disease pathogenesis remains unresolved. 7 Most of these type 2 (T2) driven diseases are accompanied with eosinophilia in the circulation. 9,10 In contrast, eosinophil numbers are markedly reduced in response to bacterial infection. 11 This phenomenon was initially believed to result from enhanced release of adrenal glucocorticoids. 12 However, J Leukoc Biol. 2020; 1-7. www.jleukbio.org 1 adrenalectomized mice subjected to acute pneumococcal infection still displayed eosinopenia in the absence of enhanced circulating corticosterone levels. 11 Therefore, the disappearance of eosinophils from the circulation must be mediated by a different mechanism. So far, this mechanism has not been elucidated and especially hard to address in humans. Understanding of the underlying mechanism might teach us more about type 2 immunity, since there is clear cross-talk between T2 immunity and innate immune responses. This is clearly illustrated by studies showing that exposure to environmental LPS can affect the clinical course of allergic diseases, including asthma. 13 In accordance, it was demonstrated that CD14, a co-receptor for LPS, is one of the risk genes for asthma. 14  was injected (t = 0 h) to induce a controlled systemic inflammatory response. Bone marrow and blood were sampled at three timepoints: at baseline (7 days before endotoxin administration), 4 h after endotoxin administration, and in 6 out of 7 volunteers 7 days after endotoxin administration ( Fig. 1).

Sample processing
Blood was collected in sodium heparin tubes (Vacuette R Greiner bio-one, Kremsmünster, Austria) and bone marrow aspirates were collected in syringes containing a sodium heparin solution (3:1 ratio).
Thereafter, erythrocytes were lysed using an ice cold isotonic lysis buffer consisting of 150 mM NH 4

Flow cytometry
One million cells were stained with antibodies for 30 min at a con-

Cytokine analysis
Cytokine concentrations were determined in one batch using a multiplex assay (Milliplex, Millipore, Billerica, MA, USA) according to the manufacturers' guidelines.

Decrease in the number of mature eosinophils in bone marrow and blood during experimental human endotoxemia
We have previously shown that the number of eosinophils in the circulation markedly decrease during experimental human endotoxemia

Effect of acute experimental inflammation on surface markers of eosinophils
To address whether endotoxin leads to activation or degranulation of eosinophils in the BM or blood, a selection of surface markers was  Fig. 3). The same was true for most surface proteins on mature eosinophils in the BM and circulation (Fig. 2). We have also measured activation markers on mature eosinophils in the circulation of 6 new donors at baseline and after endotoxemia on a fast (<25 min) and fully automated flow cytometer (Aquios, Beckman Coulter, Pasadena, CA, USA) as was described in detail before 23 in order to decrease the effects of in vitro manipulation of cells to a minimum. Again no change in expression of activation markers after endotoxemia was found ( Supplementary Fig. 4), indicating that in vitro manipulation of eosinophils did not play a significant role in our protocol.
Therefore, blood and BM eosinophils did not seem to exhibit an activated or degranulated phenotype during endotoxemia. This is surprising, as TNF-increased after endotoxin administration in blood and in the bone marrow (see Fig. 2G). 24 This cytokine can stimulate eosinophils in vitro that can lead to degranulation. 25 However, the concentration of TNF-used in vitro (100 ng/ml) was much higher than the concentration reached in vivo (0.5 ng/ml, see Fig. 2G). 24,25 On the other hand, following endotoxin administration some other markers showed slight but significant alterations. For instance, CD193 expression on mature eosinophils in the BM was attenuated after endotoxin challenge (Fig. 3A). This difference was not observed in the circulation (Fig. 3A). One week after endotoxin challenge, expression of CD193 on BM mature eosinophils returned to normal levels (Fig. 3A).
In an earlier study, we have found that CD193 is slightly up-regulated upon maturation of eosinophils in the bone marrow. 17 So, a possible explanation as to why CD193 expression is reduced after endotoxin administration is "rejuvenation" of the BM compartment, since it is plausible that only the most mature eosinophils are released into the circulation during acute inflammation. The unchanged CD193 expression on circulatory eosinophils supports this hypothesis. Additionally, we found that the expression of CD49d on only circulatory eosinophils was also attenuated after endotoxin administration (Fig. 3B). This

Effect of endotoxin administration on plasma cytokine levels
The eosinophil-specific chemokines, eotaxin 1, 2, and 3, were measured in plasma at baseline, 4 h and 1 week after experimental endotoxemia. Eotaxin 3 was not detectable at all three time points (Fig. 3C). Eotaxin 1 and 2 both increased in blood following endotoxemia, but this increase was only statistically significant for eotaxin-1 ( Fig. 3C), although eotaxin-2 showed an obvious trend (P = 0.078).
Both chemokines returned to baseline level after 1 week (Fig. 3C).
Since, we also observed a decrease in CD49d expression on circulatory eosinophils (Fig. 3B), it is tempting to speculate that systemic endotoxin challenged led to eosinophil transmigration via VLA-4 to the tissues in response to local production of eotaxin-1. 28 In contrast, L-selectin did not seem to be involved in transmigration of eosinophils as its expression did not change ( Fig. 2D and Supplementary Fig. 4C).
This is different from the role of L-selectin in neutrophils where it has been shown that LPS can activate neutrophils through this receptor. 29 The plasma levels of IL-3, IL-5, and GM-CSF were close to their detection limits in the plasma during steady state (Fig. 3D). Following 4 h and 1 week after endotoxemia, the level of these cytokines did not show evident alterations (Fig. 3D).
Our findings implicate that the eosinophil compartment is influenced by endotoxin-induced systemic inflammation beyond the Th2/ILC2-induced allergic type of response. This nonallergic mechanism that is present during endotoxin-induced innate immune responses might be important in homing of eosinophils to tissues that contain these cells also in homeostasis such as gut, 30 uterus, 31 thymus, 5 and adipose tissue. 3 It is intuitive that such homeostatic homing is not associated with cytotoxic activation of the cells as the risk for collateral damage to the tissue is too large. Similar mechanisms might also contribute to recruitment of eosinophils to tissues in allergic diseases as the gene for the LPS co-receptor CD14 is a risk gene for allergic asthma. 14