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Interleukin 7 receptor is required for myeloid cell homeostasis and reconstitution by hematopoietic stem cells

  • Taylor Cool
    Affiliations
    Institute for the Biology of Stem Cells, University of California—Santa Cruz, Santa Cruz, CA

    Program in Biomedical Sciences and Engineering, Department of Molecular, Cell, and Developmental Biology, University of California—Santa Cruz, Santa Cruz, CA
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  • Atesh Worthington
    Affiliations
    Institute for the Biology of Stem Cells, University of California—Santa Cruz, Santa Cruz, CA

    Program in Biomedical Sciences and Engineering, Department of Molecular, Cell, and Developmental Biology, University of California—Santa Cruz, Santa Cruz, CA
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  • Donna Poscablo
    Affiliations
    Institute for the Biology of Stem Cells, University of California—Santa Cruz, Santa Cruz, CA

    Program in Biomedical Sciences and Engineering, Department of Molecular, Cell, and Developmental Biology, University of California—Santa Cruz, Santa Cruz, CA
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  • Adeel Hussaini
    Affiliations
    Institute for the Biology of Stem Cells, University of California—Santa Cruz, Santa Cruz, CA
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  • E. Camilla Forsberg
    Correspondence
    Offprint requests to: E. Camilla Forsberg, Biomolecular Engineering, Baskin School of Engineering, University of California–Santa Cruz, 1156 High Street, Mail Stop SOE2 Physical Sciences, Santa Cruz, CA 95064
    Affiliations
    Institute for the Biology of Stem Cells, University of California—Santa Cruz, Santa Cruz, CA

    Biomolecular Engineering, University of California—Santa Cruz, Santa Cruz, CA
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Open ArchivePublished:September 07, 2020DOI:https://doi.org/10.1016/j.exphem.2020.09.001

      Highlights

      • Loss of IL7Rα resulted in significantly fewer eosinophils in adult mice.
      • IL7R-Cre lineage tracing revealed minimal labeling of eosinophils.
      • IL7Rα-deficient HSCs robustly reconstituted eosinophils in a WT host.
      • WT HSCs failed to fully reconstitute eosinophils in IL7Rα−/− hosts.
      • Several cytokines are differentially expressed in WT and IL7Rα-deficient mice.
      Respiratory diseases are a leading cause of death worldwide, with vulnerability to disease varying greatly between individuals. The reasons underlying disease susceptibility are unknown, but there is often a variable immune response in lungs often. Recently, we identified a surprising novel role for the interleukin 7 receptor (IL7R), a primarily lymphoid-associated regulator, in fetal-specified, lung-resident macrophage development. Here, we report that traditional, hematopoietic stem cell-derived myeloid cells in the adult lung, peripheral blood, and bone marrow also depend on IL7R expression. Using single- and double-germline knockout models, we found that eosinophil numbers were reduced on deletion of IL7Rα. We then employed two Cre recombinase models in lineage tracing experiments to test whether these cells developed through an IL7Rα+ pathway. Despite the impact of IL7Rα deletion, IL7R-Cre labeled only a minimal fraction of eosinophils. We therefore examined the intrinsic versus extrinsic requirement for IL7R in the production of eosinophils using reciprocal hematopoietic stem cell transplantation assays. These assays revealed that extrinsic, but not eosinophil-intrinsic, IL7R is required for eosinophil reconstitution by HSCs in the adult lung. To determine which external factors may be influencing eosinophil development and survival, we performed a cytokine array analysis between wild-type and IL7Rα-deficient mice and found several differentially regulated proteins. These findings expand on our previous report that IL7R is required not only for proper lymphoid cell development and homeostasis, but also for myeloid cell homeostasis in tissues.

      Graphical abstract

      Hematopoiesis is the process of mature blood and immune cell production and maintenance from hematopoietic stem and progenitor cells (HSPCs). The majority of “traditional” blood and immune cells, including eosinophils, have short half-lives and are continually replaced by HSPCs [
      • McRae H
      • Voss A
      • Thomas T
      Are transplantable stem cells required for adult hematopoiesis?.
      ,
      • Ugarte F
      • Sousae R
      • Cinquin B
      • et al.
      Progressive chromatin condensation and H3K9 methylation regulate the differentiation of embryonic and hematopoietic stem cells.
      ,
      • Smith-Berdan S
      • Nguyen A
      • Hong MA
      • Forsberg EC
      ROBO4-mediated vascular integrity regulates the directionality of hematopoietic stem cell trafficking.
      ,
      • Boyer SW
      • Rajendiran S
      • Beaudin AE
      • et al.
      Clonal and quantitative in vivo assessment of hematopoietic stem cell differentiation reveals strong erythroid potential of multipotent cells.
      ,
      • Smith-Berdan S
      • Bercasio A
      • Rajendiran S
      • Forsberg EC
      Viagra enables efficient, single-day hematopoietic stem cell mobilization.
      ,
      • Rajendiran S
      • Smith-Berdan S
      • Kunz L
      • et al.
      Ubiquitous overexpression of CXCL12 confers radiation protection and enhances mobilization of hematopoietic stem and progenitor cells.
      . In contrast, fetally derived tissue-resident macrophage (trMacs) self-maintain in their respective tissues for life, without contribution from adult HSCs, thereby leading us to consider them “nontraditional” mature immune cells [
      • Cool T
      • Forsberg EC
      Chasing Mavericks: the quest for defining developmental waves of hematopoiesis.
      ,
      • Lavin Y
      • Mortha A
      • Rahman A
      • Merad M
      Regulation of macrophage development and function in peripheral tissues.
      ,
      • Sawai CM
      • Babovic S
      • Upadhaya S
      • et al.
      Hematopoietic stem cells are the major source of multilineage hematopoiesis in adult animals.
      ,
      • Beaudin AE
      • Forsberg EC
      To B1a or not to B1a: do hematopoietic stem cells contribute to tissue-resident immune cells?.
      ]. Recently, our laboratory uncovered a novel role for interleukin 7 receptor (IL7R) in trMac specification during development [
      • Leung GA
      • Cool T
      • Valencia CH
      • Worthington A
      • Beaudin AE
      • Forsberg EC
      The lymphoid-associated interleukin 7 receptor (IL7R) regulates tissue-resident macrophage development.
      ]. Though IL7R had originally been reported to be essential exclusively for lymphocyte development and survival, a few recent studies, including ours, have found that it also plays roles in myeloid cell development [
      • Leung GA
      • Cool T
      • Valencia CH
      • Worthington A
      • Beaudin AE
      • Forsberg EC
      The lymphoid-associated interleukin 7 receptor (IL7R) regulates tissue-resident macrophage development.
      ,
      • Kelly EAB
      • et al.
      Potential contribution of IL-7 to allergen-induced eosinophilic airway inflammation in asthma.
      ,
      • Cook EB
      • Stahl JL
      • Schwantes EA
      • Fox KE
      • Mathur SK
      IL-3 and TNFα increase thymic stromal lymphopoietin receptor (TSLPR) expression on eosinophils and enhance TSLP-stimulated degranulation.
      ]. Interestingly, in addition to our finding that IL7R regulates trMac specification during mouse fetal development [
      • Sawai CM
      • Babovic S
      • Upadhaya S
      • et al.
      Hematopoietic stem cells are the major source of multilineage hematopoiesis in adult animals.
      ], studies in humans have reported that other myeloid cell types, including monocytes and eosinophils, may express IL7R [
      • Kelly EAB
      • et al.
      Potential contribution of IL-7 to allergen-induced eosinophilic airway inflammation in asthma.
      ,
      • Al-Mossawi H
      • Yager N
      • Taylor CA
      • et al.
      Context-specific regulation of surface and soluble IL7R expression by an autoimmune risk allele.
      ]. Furthermore, these cells can upregulate IL7R mRNA and surface protein on stimulation with lipopolysaccharide (LPS), a known activator of eosinophils. Here, we used germline knockout mice, lineage tracing, and transplantation assays to determine whether IL7R plays a role in adult-derived “traditional” myeloid cell types in the lung, peripheral blood (PB), and bone marrow (BM). (Please refer to the Supplementary Data [online only, available at www.exphem.org] for the Methods section of this article.)

      Results and discussion

      Adult neutrophils and eosinophils are differentially affected by deletion of IL7Rα

      During our previous studies in which we found that IL7R unexpectedly regulates trMac development, we also noted alterations in other cells types in the lungs of IL7Rα−/− mice. We therefore used Flk2−/− and IL7Rα−/− mice and crossed the two strains to also create homozygous double mutants (referred to as FIDKO mice here) to investigate lung cellularity in three mutant cohorts. Flk2 is a tyrosine kinase receptor that regulates hematopoietic development [
      • Mackarehtschian K
      • Hardin JD
      • Moore KA
      • Boast S
      • Goff SP
      • Lemischka IR
      Targeted disruption of the flk2/flt3 gene leads to deficiencies in primitive hematopoietic progenitors.
      ], including robust numbers of common myeloid progenitor cells (CMPs) [
      • Mackarehtschian K
      • Hardin JD
      • Moore KA
      • Boast S
      • Goff SP
      • Lemischka IR
      Targeted disruption of the flk2/flt3 gene leads to deficiencies in primitive hematopoietic progenitors.
      ,
      • Beaudin AE
      • Boyer SW
      • Forsberg EC
      Flk2/Flt3 promotes both myeloid and lymphoid development by expanding non-self-renewing multipotent hematopoietic progenitor cells.
      ], the presumed precursors of eosinophils. Previously, our laboratory had established that all adult-derived “traditional” mature blood and immune cells, but not trMacs [
      • Leung GA
      • Cool T
      • Valencia CH
      • Worthington A
      • Beaudin AE
      • Forsberg EC
      The lymphoid-associated interleukin 7 receptor (IL7R) regulates tissue-resident macrophage development.
      ,
      • Epelman S
      • Lavine KJ
      • Beaudin AE
      • et al.
      Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.
      ], develop through a Flk2+ pathway [
      • Leung GA
      • Cool T
      • Valencia CH
      • Worthington A
      • Beaudin AE
      • Forsberg EC
      The lymphoid-associated interleukin 7 receptor (IL7R) regulates tissue-resident macrophage development.
      ,
      • Beaudin AE
      • Boyer SW
      • Forsberg EC
      Flk2/Flt3 promotes both myeloid and lymphoid development by expanding non-self-renewing multipotent hematopoietic progenitor cells.
      ,
      • Beaudin AE
      • Boyer SW
      • et al.
      A transient developmental hematopoietic stem cell gives rise to innate-like B and T cells.
      ,
      • Boyer SW
      • Beaudin AE
      • Forsberg EC
      Mapping differentiation pathways from hematopoietic stem cells using Flk2/Flt3 lineage tracing.
      ]. As expected based on previous reports of circulating lymphocytes [
      • Sitnicka E
      • Brakebusch C
      • Martensson IL
      • et al.
      Complementary signaling through flt3 and interleukin-7 receptor α is indispensable for fetal and adult B cell genesis.
      ], B lymphocytes in the lungs were decreased in all three mutant strains relative to the wild-type (WT) controls, with drastic reductions in the IL7Rα−/− and FIDKO mice (Figure 1A), while neutrophil numbers were normal in all three genetic models (Figure 1B). Surprisingly, deletion of IL7Rα also led to a significant reduction in numbers of eosinophils in the lungs, with a further significant reduction in the FIDKO mice (Figure 1C). To determine whether this was a tissue-specific phenotype, we also analyzed the PB and BM of WT and IL7R−/− mice. We observed that eosinophils were similarly affected in these tissues (Supplementary Figure E1B,D, online only, available at www.exphem.org). Interestingly, there was no significant difference in neutrophil numbers in the BM of these mice, but there were significantly more circulating neutrophils in the IL7Rα−/− mice (Supplementary Figure E1A,C). These data indicate that IL7R is differentially required for eosinophil and neutrophil homeostasis across tissues.
      Figure 1
      Figure 1Deletion of Flk2 and/or IL7Rα differentially affects numbers of both lymphoid and myeloid cells in the lungs of adult mice. Cellularity was calculated by dividing the total numbers of cells collected by the weight of the tissue. Dots represent individual mice. Black bars represent the average. *p < 0.05, **p < 0.005, ***p < 0.001. (A) B lymphocytes were significantly reduced in Il7Rα−/− and FIDKO, but not Flk2−/−, lungs. Quantification of lymphocytes (Live, Ter119-Mac1-Gr1-CD19+) per gram of tissue in the lungs of WT (black), Flk2−/− (gray), IL7Rα−/− (white), or FIDKO (red) adult mouse lungs. WT n = 8, Flk2−/− n = 8, IL7R−/− n = 8, and FIDKO n = 5 representing four independent experiments. (B) Neutrophils were not affected by loss of Flk2 or IL7Rα. Quantification of neutrophils (Live, CD3CD4CD5CD8B220Ter119CD45+Ly6g+CD11b+) per gram of tissue in the lungs of WT (black), Flk2−/− (gray), IL7Rα−/− (white), or FIDKO (red) adult mouse lungs. WT n = 8, Flk2−/− n = 7, IL7R−/− n = 8, and FIDKO n = 6 representing four independent experiments. (C) Eosinophils were significantly reduced in Flk2−/−, IL7Rα−/−, and FIDKO mouse lungs. Quantification of eosinophils (Live,CD3CD4CD5CD8B220Ter119CD45+Ly6gCD11b+SiglecFmidCD11c) per gram of tissue in the lungs of WT (black), Flk2−/− (gray), IL7Rα−/− (white), or Flk2−/−IL7Rα−/− (FIDKO) (red) adult mouse lungs. WT n = 6, Flk2−/− n = 5, IL7R−/− n = 5, and FIDKO n = 5 representing four independent experiments.

      IL7R-Cre does not label adult neutrophils or eosinophils

      Recently, we established that IL7R-Cre, but not Flk2-Cre, robustly labels trMacs across several tissues because of transient, developmental expression of IL7Rα in the monocyte–macrophage lineage [
      • Leung GA
      • Cool T
      • Valencia CH
      • Worthington A
      • Beaudin AE
      • Forsberg EC
      The lymphoid-associated interleukin 7 receptor (IL7R) regulates tissue-resident macrophage development.
      ]. To interrogate whether eosinophils have a history of IL7R expression, we crossed mice expressing IL7R-Cre [
      • Schlenner SM
      • Madan V
      • Busch K
      • et al.
      Fate mapping reveals separate origins of T cells and myeloid lineages in the thymus.
      ] with mTmG mice containing a dual-color fluorescent reporter [
      • Muzumdar M
      • Tasic B
      • Miyamichi K
      • Li L
      • Luo L
      A global double-fluorescent Cre reporter mouse.
      ], thereby creating the “IL7RαSwitch” model analogous to the previously described FlkSwitch mouse (Figure 2A,B) [
      • Leung GA
      • Cool T
      • Valencia CH
      • Worthington A
      • Beaudin AE
      • Forsberg EC
      The lymphoid-associated interleukin 7 receptor (IL7R) regulates tissue-resident macrophage development.
      ,
      • Boyer SW
      • Schroeder AV
      • Smith-Berdan S
      • Forsberg EC
      All hematopoietic cells develop from hematopoietic stem cells through Flk2/Flt3-positive progenitor cells.
      ,
      • Boyer SW
      • Beaudin AE
      • Forsberg EC
      Mapping differentiation pathways from hematopoietic stem cells using Flk2/Flt3 lineage tracing.
      ,
      • Beaudin AE
      • Boyer SW
      • et al.
      A transient developmental hematopoietic stem cell gives rise to innate-like B and T cells.
      ]. In both models, all cells express Tomato (Tom) unless Cre-mediated recombination cause an irreversible switch to green fluorescent protein (GFP) expression by Cre-expressing cells and all of their progeny (Figure 2A,B). Labeling of lung B lymphocytes was high (>95%) in both models (Figure 2C), as has previously been reported for circulatory lymphocytes [
      • Leung GA
      • Cool T
      • Valencia CH
      • Worthington A
      • Beaudin AE
      • Forsberg EC
      The lymphoid-associated interleukin 7 receptor (IL7R) regulates tissue-resident macrophage development.
      ,
      • Schlenner SM
      • Madan V
      • Busch K
      • et al.
      Fate mapping reveals separate origins of T cells and myeloid lineages in the thymus.
      ]. Similar to myeloid cells in the PB, labeling of lung neutrophils and eosinophils was high (>95%) in the FlkSwitch mice (Figure 2D–E). In contrast, in the IL7RαSwitch mice, labeling of lung neutrophils was nominal (<3%) (Figure 2D), similar to that of previously reported PB neutrophils [
      • Leung GA
      • Cool T
      • Valencia CH
      • Worthington A
      • Beaudin AE
      • Forsberg EC
      The lymphoid-associated interleukin 7 receptor (IL7R) regulates tissue-resident macrophage development.
      ,
      • Schlenner SM
      • Madan V
      • Busch K
      • et al.
      Fate mapping reveals separate origins of T cells and myeloid lineages in the thymus.
      ]. Despite the significant reduction in eosinophil numbers in IL7Rα−/− mice (Figure 1C), labeling of eosinophils was also nominal (<3%) in the IL7RαSwitch mice (Figure 2E). These data indicate that most lung eosinophils do not develop through an IL7Rα+ pathway yet depend on IL7R for maintenance of normal cell numbers.
      Figure 2
      Figure 2IL7R-Cre efficiently labels lung B cells but does not label adult lung neutrophils or eosinophils. (A) Schematic of the “Switch” models. Cre recombinase expression was controlled by either Flk2 or IL7Rα regulatory elements. Cre-driver mice were crossed with the mTmG mice expressing a dual-color reporter expressing either Tom or GFP, under control of the Rosa26 locus. Expression of Cre results in an irreversible genetic deletion event that causes a switch in reporter expression from Tom to GFP. (B) Schematic of Cre-mediated reporter switching in the “switch” models. All cells express Tom unless expression of Cre resulted in an irreversible switch from Tomato to GFP expression. Once a cell expresses GFP, it can only give rise to GFP-expressing progeny. (C) Lung B cells were highly labeled by both Flk2-Cre and IL7R-Cre lineage tracing. Representative flow cytometric analysis of reporter expression in CD19+ lymphocytes in the lungs of adult FlkSwitch and IL7RSwitch mice. Expression of Tomato (Tom) and GFP is highlighted by red and green boxes, respectively, in FlkSwitch and IL7Rswitch models. (D) Lung neutrophils were highly labeled by Flk2-Cre, but not IL7R-Cre, lineage tracing. Representative flow cytometric analysis of reporter expression in CD45+Lin-Ly6ghi neutrophils in the lungs of adult FlkSwitch and IL7Rswitch mice. Tom/GFP gating strategies as in (C). (E) Lung eosinophils were highly labeled by Flk2-Cre, but not IL7R-Cre, lineage tracing. Representative flow cytometric analysis of reporter expression in CD45+LinLy6gloSiglecFmidCD11c eosinophils in the lungs of adult FlkSwitch and IL7Rswitch mice.
      Plots and values are representative of at least three or four mice per cohort from four independent experiments. Values indicate the mean frequency ± SEM of gated GFP+ populations.

      IL7Rα−/− HSCs efficiently generate lung eosinophils on transplantation

      To determine whether IL7R-deficient progenitors lack the intrinsic potential to make eosinophils, we transplanted IL7Rα−/− or WT HSCs into sublethally irradiated WT recipients and analyzed immune cell reconstitution in the blood and lung >4 months post transplant (Figure 3A) [
      • Ugarte F
      • Sousae R
      • Cinquin B
      • et al.
      Progressive chromatin condensation and H3K9 methylation regulate the differentiation of embryonic and hematopoietic stem cells.
      ,
      • Smith-Berdan S
      • Nguyen A
      • Hong MA
      • Forsberg EC
      ROBO4-mediated vascular integrity regulates the directionality of hematopoietic stem cell trafficking.
      ,
      • Boyer SW
      • Rajendiran S
      • Beaudin AE
      • et al.
      Clonal and quantitative in vivo assessment of hematopoietic stem cell differentiation reveals strong erythroid potential of multipotent cells.
      ,
      • Smith-Berdan S
      • Bercasio A
      • Rajendiran S
      • Forsberg EC
      Viagra enables efficient, single-day hematopoietic stem cell mobilization.
      ,
      • Rajendiran S
      • Smith-Berdan S
      • Kunz L
      • et al.
      Ubiquitous overexpression of CXCL12 confers radiation protection and enhances mobilization of hematopoietic stem and progenitor cells.
      ]. As expected, the IL7Rα−/− HSCs exhibited diminished ability to produce circulating lymphoid, but not “traditional” myeloid, cells in the blood of the same mice (Supplementary Figure E2A,B, online only, available at www.exphem.org). In contrast, lung neutrophils and eosinophils were generated with equal efficiency by WT and IL7Rα−/− HSCs (Figure 3B). The strong reconstitution potential of eosinophils and neutrophils of the lung, comparable to the GM donor chimerism in the periphery, further supports that these cells are “traditional,” adult HSC-derived myeloid cell types and that they do not intrinsically require IL7R for their development.
      Figure 3
      Figure 3IL7Rα is extrinsically required for eosinophil homeostasis in the lung. (A) Schematic depicting the transplantation experimental setup used to determine whether IL7R regulates eosinophil development by cell intrinsic mechanisms. Five hundred WT or IL7Rα−/− HSCs were transplanted into a three-fourths sublethally irradiated WT GFP recipient. After 16 weeks posttransplant, the lungs were harvested and mature immune cells were analyzed via flow cytometry for donor chimerism. (B) IL7R deletion did not alter the ability of HSCs to reconstitute lung eosinophils. Percentage donor chimerism of neutrophils and eosinophils in the lungs of transplanted mice. Error bars are SEM, WT n = 4, IL7Rα−/− n = 6, from two independent experiments. (C) Schematic depicting the transplantation experimental setup used to determine whether cell extrinsic IL7R expression is necessary for eosinophil development and homeostasis. (D) WT HSCs were less efficient at generating “traditional” myeloid cells in an IL7Rα−/− host. Percentage donor chimerism of neutrophils and eosinophils in the lungs of transplanted mice. Error bars are SEM, WT n = 5, IL7Rα−/− n = 13, from four independent experiments. *p < 0.05, **p < 0.005, ***p < 0.001. (E) Loss of IL7R results in altered cytokine profile in adult mice. Dot plots represent the protein concentration (in pg/mL) of WT (black dots) or IL7Rα−/− (white dots) in the serum of adult mice. Black bars represent the average. WT n = 3, IL7Rα−/− n = 3, from three independent mice; this was sufficient to reach a statistical power of 80% and *p < 0.05. (F) Flk2 and IL7R are differentially involved in the development of multiple hematopoietic cell types. Schematic depicting the differential expression and functional requirement for IL7R and Flk2 in traditional lymphocytes (B cells, top), tissue-resident macrophages (trMacs, middle), and adult lung myeloid cells (eosinophils, bottom). Lymphocytes are highly labeled by both Flk2-Cre (FlkSwitch) and IL7R-Cre (IL7RαSwitch), and functionally dependent on both receptors, although more drastic reductions in cell numbers were observed in IL7Rɑ−/− (thick arrow) than in Flk2−/− (thin arrow) mice. In contrast, trMacs are highly labeled by IL7R-Cre, but not Flk2-Cre, and functionally rely on IL7R (thin arrow), but not Flk2 (black line), for efficient development. Here, we report that lung eosinophils are highly labeled by Flk2-Cre, but not IL7R-Cre, yet depend on IL7R (thick arrow), but not Flk2 (black line), for efficient maintenance and reconstitution by HSCs.

      Wild-type HSCs fail to fully reconstitute eosinophils in IL7Rα−/− recipients

      To determine whether the requirement for IL7R was eosinophil extrinsic, WT HSCs were transplanted into either WT or IL7Rα−/−recipients (Figure 3C). WT HSCs efficiently contributed to circulating GMs, B cells, and T cells (Supplementary Figure E2C,D), but, intriguingly, displayed significantly impaired reconstitution of neutrophils and eosinophils in the lungs of IL7Rα−/− recipients (Figure 3D). Taken together, these data suggest that extrinsic IL7R is required for homeostasis of neutrophils and eosinophils in the lung, but not for that of other “traditional” myeloid cells that circulate in the periphery.

      IL7R deletion caused alterations in cytokines involved in myeloid cell homeostasis

      Previous studies have reported several factors implicated in eosinophil development, homing, and survival, including eotaxin [
      • Saito H
      • Honda K
      • Asaka C
      • Ueki S
      • Ishikawa K
      Eosinophil chemotaxis assay in nasal polyps by using a novel optical device EZ-TAXIScan: role of CC-chemokine receptor 3.
      ,
      • Rose Jr, CE
      • Lannigan JA
      • Kim P
      • Lee JJ
      • Fu SM
      • Sung SSJ
      Murine lung eosinophil activation and chemokine production in allergic airway inflammation.
      ], IL-5 [
      • Wiesner DL
      • Smith KD
      • Kashem SW
      • Bohjanen PR
      • Nielsen K
      Different lymphocyte populations direct dichotomous eosinophil or neutrophil responses to pulmonary Cryptococcus infection.
      ,
      • Walker C
      • Checkel J
      • Cammisuli S
      • Leibson PJ
      • Gleich GJ
      IL-5 production by NK cells contributes to eosinophil infiltration in a mouse model of allergic inflammation.
      ,
      • Foster PS
      • Hogan SP
      • Ramsay AJ
      • Matthaei KI
      • Young IG
      Interleukin 5 deficiency abolishes eosinophilia, airways hyperreactivity, and lung damage in a mouse asthma model.
      ,
      • Takatsu K
      Interleukin-5 and IL-5 receptor in health and diseases.
      ], and others [
      • Kelly EAB
      • et al.
      Potential contribution of IL-7 to allergen-induced eosinophilic airway inflammation in asthma.
      ,
      • Cook EB
      • Stahl JL
      • Schwantes EA
      • Fox KE
      • Mathur SK
      IL-3 and TNFα increase thymic stromal lymphopoietin receptor (TSLPR) expression on eosinophils and enhance TSLP-stimulated degranulation.
      ,
      • Yi S
      • Zhai J
      • Niu R
      • et al.
      Eosinophil recruitment is dynamically regulated by interplay among lung dendritic cell subsets after allergen challenge.
      ]. To determine which factors may be influencing eosinophil development and survival in the IL7R mutant mice, we collected serum from WT and mutant mice and compared the relative concentrations of several cytokines. Because IL-7 has previously been reported to be elevated in IL7Rα−/− mice [
      • Kelly EAB
      • et al.
      Potential contribution of IL-7 to allergen-induced eosinophilic airway inflammation in asthma.
      ], we first tested IL-7 levels as our positive control. Consistent with previous data, we observed that IL-7 was upregulated severalfold in IL7Rα−/− mice, although borderline statistically significant here (∼10-fold, p < 0.1; Figure 3E). Importantly, we also observed that eosinophil-promoting eotaxin was significantly downregulated in the IL7Rα−/− mice (Figure 3E). Additionally, IL-5 was decreased threefold in the mutant mice, near statistical significance (p < 0.1; Figure 3E). Taken together, these data indicate that downregulation of eotaxin and IL-5, and possibly additional factors, may play important roles in eosinophil development and survival in the IL7R mutant mice.
      Our data described here reveal that IL7R is required for specific myeloid cell homeostasis in the lungs, PB, and BM of adult mice. Other groups have reported that human eosinophils have detectable levels of IL7Rα mRNA and surface protein [
      • Kelly EAB
      • et al.
      Potential contribution of IL-7 to allergen-induced eosinophilic airway inflammation in asthma.
      ,
      • Cook EB
      • Stahl JL
      • Schwantes EA
      • Fox KE
      • Mathur SK
      IL-3 and TNFα increase thymic stromal lymphopoietin receptor (TSLPR) expression on eosinophils and enhance TSLP-stimulated degranulation.
      ] and that IL7R mRNA can be induced in human monocytes with LPS stimulation [
      • Al-Mossawi H
      • Yager N
      • Taylor CA
      • et al.
      Context-specific regulation of surface and soluble IL7R expression by an autoimmune risk allele.
      ]. Similarly, we previously found that trMacs in the lung and other tissues transiently express IL7Rα during development [
      • Leung GA
      • Cool T
      • Valencia CH
      • Worthington A
      • Beaudin AE
      • Forsberg EC
      The lymphoid-associated interleukin 7 receptor (IL7R) regulates tissue-resident macrophage development.
      ]. However, our IL7R-Cre lineage tracing data of traditional lung myeloid cells reported here indicate that only a small proportion of eosinophils and neutrophils are labeled at steady state. This strongly argues against a requirement for expression by eosinophils or their precursors. Instead, we favor a model of cell-extrinsic requirement for IL7Rα in eosinophil development. This notion is supported by the reciprocal transplantation assays, where we found that WT and IL7Rα−/− HSCs are equally capable of contributing to eosinophils and neutrophils in WT hosts (Figure 3B). Conversely, when we transplanted WT HSCs into an IL7R null background, we observed that the donor chimerism of eosinophils and neutrophils in the lungs was significantly impaired (Figure 3D), but that circulating “traditional” myeloid cells were efficiently generated (Supplementary Figure E2B). Interestingly, it has been reported that eosinophil homeostasis relies on lymphocyte- and stroma-secreted survival factors [
      • Wiesner DL
      • Smith KD
      • Kashem SW
      • Bohjanen PR
      • Nielsen K
      Different lymphocyte populations direct dichotomous eosinophil or neutrophil responses to pulmonary Cryptococcus infection.
      ,
      • Walker C
      • Checkel J
      • Cammisuli S
      • Leibson PJ
      • Gleich GJ
      IL-5 production by NK cells contributes to eosinophil infiltration in a mouse model of allergic inflammation.
      ,
      • Yi S
      • Zhai J
      • Niu R
      • et al.
      Eosinophil recruitment is dynamically regulated by interplay among lung dendritic cell subsets after allergen challenge.
      ,
      • Abdala-Valencia H
      • Coden ME
      • Chiarella SE
      • et al.
      Shaping eosinophil identity in the tissue contexts of development, homeostasis, and disease.
      ,
      • Rochman Y
      • Leonard WJ
      Thymic stromal lymphopoietin: a new cytokine in asthma.
      ,
      • Hogan MB
      • Weissman DN
      • Hubbs AF
      • Gibson LF
      • Piktel D
      • Landreth KS
      Regulation of eosinophilopoiesis in a murine model of asthma.
      ]. We found that two known eosinophil regulators, eotaxin and IL-5, were reduced in the IL7Rα−/− mice. Both of these cytokines are known to be secreted by lymphoid cells to promote eosinophil development, homing, and survival [
      • Saito H
      • Honda K
      • Asaka C
      • Ueki S
      • Ishikawa K
      Eosinophil chemotaxis assay in nasal polyps by using a novel optical device EZ-TAXIScan: role of CC-chemokine receptor 3.
      ,
      • Rose Jr, CE
      • Lannigan JA
      • Kim P
      • Lee JJ
      • Fu SM
      • Sung SSJ
      Murine lung eosinophil activation and chemokine production in allergic airway inflammation.
      ,
      • Walker C
      • Checkel J
      • Cammisuli S
      • Leibson PJ
      • Gleich GJ
      IL-5 production by NK cells contributes to eosinophil infiltration in a mouse model of allergic inflammation.
      ,
      • Foster PS
      • Hogan SP
      • Ramsay AJ
      • Matthaei KI
      • Young IG
      Interleukin 5 deficiency abolishes eosinophilia, airways hyperreactivity, and lung damage in a mouse asthma model.
      ,
      • Takatsu K
      Interleukin-5 and IL-5 receptor in health and diseases.
      ,
      • Hartnell A
      • Robinson DS
      • Kay AB
      • Wardlaw AJ
      CD69 is expressed by human eosinophils activated in vivo in asthma and in vitro by cytokines.
      ]. Additionally, as previously reported [
      • Kelly EAB
      • et al.
      Potential contribution of IL-7 to allergen-induced eosinophilic airway inflammation in asthma.
      ], we observed that IL-7 was upregulated in the IL7R−/− mice, likely because of unbound excess IL-7 in the absence of lymphoid cells. These data could potentially explain the mechanisms underlying the eosinophil decrease in IL7Rα−/− mice (Figures 1C and 3D; Supplementary Figure E1B,D), which have drastically reduced numbers of lymphocytes [
      • Boyer SW
      • Beaudin AE
      • Forsberg EC
      Mapping differentiation pathways from hematopoietic stem cells using Flk2/Flt3 lineage tracing.
      ] (Figure 1A; Supplementary Figure E2B,D). The lack of lymphoid cells results in less secretion of eotaxin and IL-5, resulting in poor eosinophil development and survival. This suggests an extrinsic requirement for IL7R for eosinophil and neutrophil homeostasis. The data reported here represent a novel role of IL7R in “traditional” myeloid cell homeostasis, in a cell type-specific manner. We interpret our findings to suggest a cell-intrinsic role for IL7R in lymphoid cell development and survival, which then, in turn, supports traditional myeloid cells in the lungs of adult mice. The results presented here add to the body of knowledge of how complex and dynamic Flk2 and IL7R expression and function cooperate in the regulation of a variety of immune cells, including traditional myeloid cells in the adult lung (Figure 3F). These findings are significant for lung health, because understanding hematopoietic homeostasis in the lung provides insight into susceptibility to respiratory disease.
      Supplemental Figure E1
      Supplemental Figure E1Neutrophil and eosinophil homeostasis are altered in the bone marrow (BM) and peripheral blood (PB) of IL7Rα−/− mice. Quantification of total cells per 25ul of PB or 1 leg (for BM) of WT (black) or IL7Rα−/− (white) adult mice. Error bars are SEM, WT n=3 and IL7Rα−/− n=4 representing two independent experiments. *P<0.05, **P<0.005, ***P<0.001.
      A, Neutrophil numbers were not significantly different in the BM of Il7Rα−/− mice. Quantification of neutrophils (Live, CD3-CD4-CD5-CD8-B220-Ter119-CD45+Ly6g+CD11b+) per 1 leg of wild type (WT) (black) or IL7Rα−/- (white) adult mice.
      B, Eosinophil numbers were significantly reduced in the BM IL7Rα−/− mice. Quantification of eosinophils (Live, CD3-CD4-CD5-CD8-B220-Ter119-CD45+Ly6g-CD11b+SiglecFmidCD11c-) per 1 leg of wild type (WT) (black) or IL7Rα−/- (white) adult mice.
      C, Neutrophil numbers were significantly increased in the PB of IL7Rα−/− mice. Quantification of neutrophils (Live, CD3-CD4-CD5-CD8-B220-Ter119-CD45+Ly6g+CD11b+) per 25ul of wild type (WT) (black) or IL7Rα−/- (white) adult blood.
      D, Eosinophil numbers were significantly reduced in the PB of IL7Rα−/− mice. Quantification of eosinophils (Live,CD3-CD4-CD5-CD8-B220-Ter119-CD45+Ly6g-CD11b+SiglecFmidCD11c-) per 25ul of wild type (WT) (black) or IL7Rα−/- (white) adult blood.
      Supplemental Figure E2
      Supplemental Figure E2A, Schematic depicting the transplantation experimental setup used to determine cell extrinsic mechanisms regulating “traditional” mature myeloid and lymphoid cell development. 500 WT or IL7Rɑ−/− HSCs were transplanted into a ¾ sublethally irradiated wild type (WT) GFP recipient. After 16 weeks post transplant, the peripheral blood was harvested and mature immune cells were analyzed via flow cytometry for donor chimerism.
      B, IL7R deletion did not alter the ability of HSCs to reconstitute “traditional” circulating myeloid cells, but did display reduced capacity to generate B and T lymphocytes. Percent donor chimerism of granulocyte/macrophage (GM) (Gr1+CD11b+), B lymphocytes (B220+), and T lymphocytes (CD3+) in the peripheral blood of the same transplanted mice as in B. Error bars are SEM, WT n = 5 IL7Rɑ−/− n=6 from 2 independent experiments. ** = p<0.05, **P<0.005, ***P<0.001.
      C, A schematic depicting the transplantation experimental setup used to determine cell extrinsic mechanisms regulating “traditional” circulating myeloid and lymphoid cells.
      D, WT HSCs robustly reconstituted “traditional” circulating myeloid and lymphoid cells in an IL7Rα−/− recipient. Percent donor chimerism of granulocyte/macrophage (GM) (Gr1+CD11b+), B lymphocytes (B220+), and T lymphocytes (CD3+) in the peripheral blood of the same transplanted mice as in D. Error bars are SEM, WT n = 5, IL7Rɑ−/− n=13 from 4 independent experiments. * = p<0.05, **P<0.005, ***P<0.001.

      Acknowledgments

      We thank Dr I Lemischka for the Flk2−/− mice; Drs H-R Rodewald and SM Schlenner for the IL7Rα-Cre strain; Dr T Boehm for the Flt3Cre strain; Bari Nazario and the UCSC Institute for the Biology of Stem Cells for flow cytometry support.
      This work was supported by an National Institutes of Health (NIH) /National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) award ( R01DK100917 ) and an American Asthma Foundation (AAF) Research Scholar award to ECF; by California Institute for Regenerative Medicine (CIRM) - Stem Cell Internships in Laboratory-based Learning (SCILL) grant TB1-01195 to TC via San Jose State University; by Tobacco-Related Disease Research Program (TRDRP) Predoctoral Fellowships to TC and AW; by American Heart Association and Howard Hughes Medical Institute Gilliam Fellowships to DP, and by CIRM Facilities awards CL1-00506 and FA1-00617-1 to University of California Santa Cruz (UCSC).

      Supplemental Materials

      Methods

      Mice

      All animals were housed and bred in the AALAC accredited vivarium at UC Santa Cruz and group housed in ventilated cages on a standard 12:12 light cycle. All procedures were approved by the UCSC Institutional Animal Care and Use (IACUC) committees. IL7Rα-Cre6,16 and Flk2-Cre (Benz et al 2008) mice, obtained under fully executed Material Transfer Agreements, were crossed to homozygous Rosa26mTmG females17 to generate “switch” lines, all on the C57Bl/6 background. WT C56Bl/6 mice were used for controls and for all expression experiments. Adult male and female mice were used randomly and indiscriminately, with the exception of FlkSwitch mice, in which only males were used because of more uniformly high floxing in male than in female mice.

      Tissue and cell isolation

      Mice were sacrificed by CO2 inhalation. Adult peripheral blood was collected by femoral artery knick. Lungs were dissected, manually dissociated, and incubated in 1x PBS(+/+) with 2% serum, 1-2mg/ml collagenase IV (Gibco) with 100U/ml Dnase1 for 1- 2 hours. Following incubation, all tissues were passed through a 16g needle ∼10X followed by a 19g needle ∼10X to make a single cell suspension, and then filtered through a 70 µM filter to obtain a single cell suspension. Cells were pelleted by centrifugation (1200g/ 4 degrees C /5 minutes). Numbers neutrophils, eosinophils, and B lymphocytes were analyzed and compared from the same tissue preparations from the same mice.

      Flow Cytometry

      Cell labeling was performed on ice in 1X PBS with 5 mM EDTA and 2% serum6. Analysis was performed on a customized BD FACS Aria II and analyzed using FlowJo. Antibodies used: cd45.2-PB (BioLegend-109820), Ter119-PGP (BioLegend-116202), CD3-PGP (BioLegend-100202), CD4-PGP (BioLegend-100402), CD5-PGP (BioLegend-100602), CD8-PGP (BioLegend-100702), B220-PGP (BioLegend-103202), CD11b-PGP (BioLegend-101202), Gr1-PGP (BioLegend-108402), Ly6g-APC (BiolLegend-127614), CD11b-PeCy7 (BiolLegend-101216), SiglecF-BV786 (BioLegend-740956), CD11c-APC-Cy7 (BioLegend-117323), B220-APC Cy7 (BioLegend-103224), CD45.2-A700 (Biolegend-109822), CD19-BV786 (Biolegend-115543), GAR-PE Cy5 (Life Technologies- A-10691).

      Transplantation Assays

      Transplantation assays were performed as previously described11,18,30–34. Briefly, sorted cells were isolated from wild type (WT) or IL7Rα knockout mice (IL7Rα−/−) donor bone marrow. WT recipient mice aged 8-12 weeks were sublethally irradiated (750 rad, single dose) using a Faxitron CP-160 (Faxitron). Under isofluorane-induced general anesthesia, sorted cells were transplanted retro-orbitally.

      Cytokine Analysis

      Serum was harvested from wild type or IL7R−/- mice via cardiac puncture. Several hundred microliters of blood was collected and transferred to a 1.5ml anti-coagulant free tube and allowed to clot at room temp for 30 min. Blood was centrifuged at 3,000 rpm for 10 min at 4C. Serum was carefully transferred to a fresh tube and stored at -80C until shipment to Eve Technologies (Calgary, Canada) for analysis..

      Quantification and Statistical Analysis

      Number of experiments, n, and what n represents can be found in the legend for each figure. Statistical significance was determined by two-tailed unpaired student's T-test. All data are shown as mean ± standard error of the mean (SEM) representing at least two to three independent experiments. Power calculations for cytokine analysis indicated that 3 mice was sufficient to achieve a statistical power of 80% at 0.9-0.95 confidence level (https://epitools.ausvet.com.au/twomeanstwo).

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