Key Points
HoxB8 progenitor–derived neutrophils can repopulate irradiated mice.
HoxB8 chimeras are useful for the in vivo analysis of neutrophil functions.
Visual Abstract
Abstract
Although neutrophils play important roles in immunity and inflammation, their analysis is strongly hindered by their short-lived and terminally differentiated nature. Prior studies reported conditional immortalization of myeloid progenitors using retroviral expression of an estrogen-dependent fusion protein of the HoxB8 transcription factor. This approach allowed the long-term culture of mouse myeloid progenitors (HoxB8 progenitors) in estrogen-containing media, followed by differentiation toward neutrophils upon estrogen withdrawal. Although several reports confirmed the in vitro functional responsiveness of the resulting differentiated cells (HoxB8 neutrophils), little is known about their capacity to perform in vivo neutrophil functions. We have addressed this issue by an in vivo transplantation approach. In vitro–generated HoxB8 neutrophils showed a neutrophil-like phenotype and were able to perform conventional neutrophil functions, like respiratory burst, chemotaxis, and phagocytosis. The i.v. injection of HoxB8 progenitors into lethally irradiated recipients resulted in the appearance of circulating donor-derived HoxB8 neutrophils. In vivo–differentiated HoxB8 neutrophils were able to migrate to the inflamed peritoneum and to phagocytose heat-killed Candida particles. The reverse passive Arthus reaction could be induced in HoxB8 chimeras but not in irradiated, nontransplanted control animals. Repeated injection of HoxB8 progenitors also allowed us to maintain stable circulating HoxB8 neutrophil counts for several days. Injection of arthritogenic K/B×N serum triggered robust arthritis in HoxB8 chimeras, but not in irradiated, nontransplanted control mice. Taken together, our results indicate that HoxB8 progenitor–derived neutrophils are capable of performing various in vivo neutrophil functions, providing a framework for using the HoxB8 system for the in vivo analysis of neutrophil function.
Introduction
Neutrophils are the most abundant circulating leukocytes in humans and a prominent leukocyte population in experimental mice (1–3). Neutrophils form the first line of defense against invading bacterial and fungal pathogens. However, their inappropriate activation may also contribute to the pathogenesis of various human diseases, including autoimmune and inflammatory diseases and cancer progression (3, 4). Based on their role in the pathogenesis of diverse human diseases, pharmacological manipulation of neutrophils has been proposed to be a potentially promising therapeutic strategy (3).
Neutrophils are short-lived cells that are believed to only survive for a short period of time in the circulation, with estimates ranging from ∼8 h to up to 5 d (3, 5). Their short lifespan is likely related to a prominent intrinsic proapoptotic program, which is kept under control by antiapoptotic molecules, with a predominant role for Mcl-1 (6, 7). Neutrophils are also terminally differentiated cells that fail to proliferate and have a transcriptionally mostly silent genetic machinery. Those features make it impossible to culture neutrophils, hindering their long-term maintenance, expansion, or genetic manipulation under in vitro conditions.
The discrepancy between the major interest in neutrophil function from biological, medical, and pharmacological perspectives and their inaccessibility by conventional tissue culture and genetic manipulation techniques has been the most important obstacle of neutrophil biology and has strongly hindered scientific progress in the field. Indeed, although primary human neutrophils can be isolated relatively easily from the human peripheral blood for short-term experiments, our tools for more complex interventions are very limited. Although there are a number of myeloid leukemia cell lines that can be differentiated toward the neutrophil lineage (8, 9), those cell lines lack several critical features of mature neutrophils (e.g., the development of various granule populations); therefore, they cannot be considered near-primary, neutrophil-like cells. In addition, their function cannot be tested in vivo either because of their malignant nature and their mostly human origin, which is poorly compatible with studies in experimental mice. The most widely used approach to test genetically manipulated neutrophils is the analysis of neutrophils from transgenic mice carrying germline mutations. Although this is a viable approach, the scope of those studies is limited by the small amount of neutrophil biological material that can be obtained and the major costs and efforts of generating and maintaining germline mutations. In addition, it is mostly impossible to influence the neutrophil developmental program of those transgenic mice; this is why any mutations need to be introduced in an early developmental (e.g., germline) stage to allow the mutation to be present in mature neutrophils (i.e., mutations cannot be introduced in the middle of the neutrophil developmental process).
Hox-family transcription factors are critical for various stages of hematopoiesis, and their excessive activation contributes to immortalization and arrested differentiation of hematopoietic cells in various forms of acute leukemia (10). In particular, overexpression of HoxA9 has been observed in more than 50% of acute myeloid leukemia and is associated with poor prognosis (11). Although HoxB8 (also known as Hox-2.4), another Hox transcription factor, is not associated with human leukemia, its deletion from the hematopoietic compartment leads to microglia-related behavioral changes in mice (12), suggesting a possible role for HoxB8 in the hematopoietic compartment. Importantly, proviral integration leading to rearrangement and overexpression of the HoxB8-encoding gene was observed in WEHI-3 cells, a mouse myelomonocytic leukemia cell line (13, 14), suggesting a possible link between HoxB8 overexpression and immortalization of myeloid cells. Follow-up experiments revealed that overexpression of HoxB8 in bone marrow cells results in the emergence of strongly IL-3–dependent myeloid cell lines that proliferate in the presence of high concentrations of IL-3 but undergo myeloid differentiation upon IL-3 withdrawal (15, 16). HoxB8 overexpression also led to specific blockade of granulocytic differentiation (17). Those results suggested that HoxB8 overexpression combined with high IL-3 concentration may immortalize myeloid progenitors and block further granulocytic differentiation, whereas IL-3 removal may lead to further differentiation, which in the absence of HoxB8 expression, may be skewed to the granulocytic lineage.
The above experiments prompted the development of an elegant approach to address the limitations of culturing and manipulating the neutrophil lineage by conditional expression of HoxB8 in myeloid progenitors (18). Retroviral expression of an ER–HoxB8 fusion protein in mouse bone marrow cells led to the generation of supposedly polyclonal myeloid progenitor cell lines that showed practically unlimited in vitro proliferation and self-renewal in the presence of estrogen (as a means of HoxB8 activation) but were able to differentiate toward neutrophils upon estrogen withdrawal (deactivation of HoxB8) and addition of G-CSF (18). This system allowed the reversible temporary immortalization of myeloid progenitors (so-called HoxB8 progenitors) and their in vitro expansion and manipulation, followed by their in vitro differentiation toward neutrophil-like cells (so-called HoxB8 neutrophils) that phenotypically and functionally resembled primary mouse neutrophils (18). Alternative protocols also yielded generation of monocytes/macrophages, dendritic cells, basophils, osteoclasts, and even lymphoid cells from conditionally immortalized progenitors using a conceptually similar approach (18–24).
HoxB8 neutrophils have been shown to be able to perform a number of in vitro functions typical for freshly isolated normal neutrophils, including reactive oxygen species production, degranulation, chemotaxis, chemokine/cytokine release, antimicrobial responses, or apoptosis, and to respond to activation through a number of cell surface receptors such as GPCR-coupled chemoattractant receptors, integrins, and Fc receptors (25–30). However, there is very limited information about their in vivo functional responsiveness. The few sporadic studies mainly focused on the in vivo appearance of HoxB8 neutrophils upon injection of HoxB8 progenitors or neutrophils into live mice and/or used approaches in which normal and HoxB8 neutrophils were simultaneously present in vivo, hindering the analysis of the functional importance of the HoxB8 progenitor–derived cells (20, 26, 31–33).
The paucity of information related to the in vivo functions of HoxB8 neutrophils prompted us to transplant HoxB8 progenitors into lethally irradiated recipients, with the aim of testing the repopulation of the neutrophil compartment by HoxB8 progenitor–derived cells and of subjecting those chimeras to various infection-related or inflammatory stimuli. Our results indicate that it is possible to generate chimeras with exclusively HoxB8 progenitor–derived neutrophils in the circulation even for a prolonged (1–2 wk) period and that those chimeras are able to undergo various in vivo inflammatory processes with the active contribution from the donor-derived HoxB8 neutrophils.
Materials and Methods
Animals and hematopoietic cell transplantation
Wild-type C57BL/6 and NOD mice as well as a congenic (B6.SJL-Ptprca) strain carrying the CD45.1 allele on the C57BL/6 genetic background were purchased from The Jackson Laboratory. Mice carrying the KRN TCR transgene (34) were obtained from D. Mathis and C. Benoist (Harvard Medical School, Boston, MA), maintained in heterozygous form by mating with C57BL/6 mice, and genotyped by allele-specific PCR. KRN-transgenic mice were then mated with NOD mice to obtain K/B×N (arthritic) and B×N (nonarthritic control) F1 offspring as described (35, 36). Mice with a ubiquitous expression of a membrane-targeted tdTomato fluorescent protein [carrying the Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo allele] (37) was obtained from The Jackson Laboratory.
Mice were kept in individually sterile ventilated cages (Tecniplast) either in a specific pathogen-free facility or an adjacent conventional facility. Experiments were approved by the Animal Experimentation Review Board of Semmelweis University. Mice of both genders at 2–6 mo of age were used for the experiments.
To generate HoxB8 chimeras, recipients carrying the CD45.1 allele on the C57BL/6 genetic background were irradiated by 11 (lethal irradiation) or 7 Gy (sublethal irradiation) from a 137Cs source using a Gamma-Service Medical D1 irradiator, followed by single or repeated i.v. injection of 3 × 107 in vitro–cultured HoxB8 progenitors per recipient at the indicated time points. Alternatively, chimeras were generated by i.v. injection of 3 × 106 unfractionated bone marrow cells per recipient (referred to as bone-marrow chimeras) or by the combination of both donor cell types (3 × 106 bone-marrow cells and 3 × 107 HoxB8 progenitors; referred to as mixed bone-marrow chimeras). To distinguish recipient cells and both donor cell types in mixed bone-marrow chimeras, bone marrow cells from mice with a ubiquitous expression of a membrane-targeted tdTomato fluorescent protein were used as bone marrow donors.
Generation and in vitro culture of HoxB8 cells
Stem cell factor (SCF)–producing Chinese hamster ovary (CHO) cells were a kind gift from H. Häcker (St. Jude Children’s Research Hospital). These cells were cultured in RPMI 1640 (Biosera) supplemented with 10% FCS (Biosera) and 100 μg/ml penicillin/streptomycin (Sigma-Aldrich).
The immortalized HoxB8 progenitor cells were generated as previously described (18, 28). Briefly, HEK-293T cells were transfected with 10 μg pMSCVneo–ER–Hoxb8 retroviral and 10 μg pCL-Eco packaging vectors (18g, 30 min at room temperature). Progenitor cells collected from the gradient interface were cultured in RPMI 1640 supplemented with 10% FCS, 100 μg/ml penicillin/streptomycin, 10 ng/ml murine IL-3 (PeproTech), 20 ng/ml murine IL-6 (PeproTech), and 2% conditioned medium of SCF-producing CHO cells. After 72 h, the progenitor cells were transduced with the retrovirus by spinoculation (1500 × g
HoxB8 progenitors used for the experiments were derived from five independent retroviral transductions, three of them using bone marrow and two of them using fetal liver cells. No phenotypical or functional difference between HoxB8 cells from the two different sources were observed (data not shown).
To trigger neutrophilic differentiation of HoxB8 progenitors, β-estradiol was withdrawn and washed out from the HoxB8 progenitor culture, and the cells were further cultured in RPMI 1640 supplemented with 10% FCS, 100 μg/ml penicillin/streptomycin, 20 ng/ml mouse G-CSF (PeproTech), 2% SCF-producing CHO cell supernatant, and 30 μM 2-ME typically for 5 d.
Flow cytometry
Blood samples were obtained from tail vein incision, washed, stained, and then resuspended in BD Biosciences’ FACS Lysing Solution. Bone marrow and spleen cell samples were obtained by flushing the bone marrow or crushing the spleen through a 70-μm cell strainer, followed by RBC lysis with RBC Lysis Buffer (eBioscience), staining, and resuspension in PBS containing 5% FCS. Samples were kept at 4°C during the entire procedure. Specified volumes were used throughout, allowing a precise determination of absolute cell counts.
Flow cytometry was performed using a BD Biosciences FACSCalibur and analyzed by FCS Express 6 Flow (De Novo Software).
RBC and platelet counts were measured by Vet-Med-Labor veterinary diagnostic services.
Cytospin and microscopy
In vitro functional assays
All in vitro functional assays were performed at 37°C in HBSS supplemented with 20 mM HEPES (pH 7.4).
Adherent respiratory burst was measured by a cytochrome C reduction test as described previously (38) using 5 × 105 HoxB8 cells in 100 μl per well in a Labsystems Multiskan Ascent multiplate reader in dual wavelength (550 and 540 nm) kinetic measurement mode. One hundred nanomolars PMA (Sigma-Aldrich) or immobilized HSA–anti-HSA immune complexes were used as stimulus as described (38, 39). For the sake of presentation, unstimulated control values were subtracted from those of stimulated samples.
For in vitro migration experiments, Transwell inserts with a polycarbonate membrane with 5-μm pore size (Corning) were precoated with fibrinogen as described (38) and filled with HoxB8 cell suspensions. The inserts were placed into 24-well plates filled with the indicated concentrations of fMLF (Sigma-Aldrich) or MIP-2 (PeproTech). After 60 min, the plates were spun, the inserts were removed, and the number of neutrophils in the bottom of the wells was determined by an acid phosphatase assay (38). Parallel samples were included to determine the signal intensity from the total cell number loaded into the Transwell inserts.
The GFP-expressing USA300 Staphylococcus aureus strain was a kind gift from W. Nauseef (University of Iowa). Heat-killed Candida albicans cells (SC5314 strain) labeled with Alexa Fluor 488 were a kind gift from A. Gácser (University of Szeged). The concentration of the bacterial or fungal cells was adjusted to an optical density at 600 nm of 1/cm in HBSS and opsonized by the addition of 500 μl normal murine serum to 800 μl microbial suspension for 30 min at 37°C. The cells were then centrifuged (5 min at 3000 × g at 4°C) and washed once in HBSS.
For the analysis of in vitro phagocytosis, 1 × 106/ml neutrophils were incubated with 1 × 107/ml nonopsonized or opsonized bacteria or fungi for 120 min at 37°C in a linear shaker (300 rpm) with samples taken every 60 min. Samples were then diluted 5-fold in ice-cold PBS containing 5% FCS and analyzed by flow cytometry. Four micrograms per milliliter trypan blue was added immediately before flow cytometry to exclude cell-associated bacteria that have not been internalized.
Thioglycollate-induced peritonitis
Peritonitis was induced by i.p. injection of 1 ml 3% thioglycollate (Liofilchem) or PBS. After the indicated time, mice were sacrificed, and the peritoneum was flushed by 5 ml ice-cold PBS containing 5% FCS. The lavage samples were washed, resuspended in PBS containing 5% FCS, and maintained at 4°C until staining for flow cytometry.
In vivo phagocytosis
7 heat-killed and Alexa Fluor 488–stained C. albicans suspended in 100 μl PBS. Sixty minutes later, the mice were sacrificed, and peritoneal lavage and staining was done as described previously. To prove that cell-associated fungi are internalized (and not only bound to the surface), 4 μg/ml trypan blue was added immediately before the flow cytometric analysis.
Reverse passive Arthus reaction
The cutaneous reverse passive Arthus reaction was triggered as described (40). Mice were anesthetized by isoflurane inhalation (Baxter Hungary), and one of the ears was injected intradermally with 20 μl rabbit polyclonal anti-OVA whole serum (Sigma-Aldrich) diluted 2-fold in PBS. Similarly prepared normal rabbit serum (Sigma-Aldrich) was injected into the other ear as control. The mice were then immediately injected i.v. with 0.8 mg OVA (Sigma-Aldrich), followed by the i.v. injection of 1 mg Evans blue 2 h later. The ear thickness before the rabbit serum injection and before Evans blue injection was measured by a spring-loaded caliper (Koeplin). Thirty minutes after Evans blue injection, the mice were sacrificed, and their ears were collected, cut into small pieces, and digested with the Liberase II kit (Roche Diagnostics) on an Eppendorf Thermomixer at 1400 rpm for 1 h at 37°C according to the manufacturer’s instructions. Single-cell suspensions were then obtained by passing the digests through a 70-μm cell strainer (BD Biosciences). The samples were then centrifuged (5000 × g, 3 min, 4°C), the pellet was stained and analyzed by flow cytometry and the concentration of MIP-2 and LTB440, 41).
K/B×N serum–transfer arthritis
Serum from KRN transgene-positive (arthritic) K/B×N and transgene-negative (nonarthritic) B×N mice was obtained as described (35, 36). Arthritis was induced by i.p. injection of 300 μl K/B×N (arthritic) or B×N (control) serum, followed by daily scoring of clinical signs of arthritis and measurement of ankle thickness for 10 d as described (35, 36).
Presentation of the data and statistical analysis
Experiments were performed the indicated number of times. Quantitative graphs and kinetic curves show mean and SEM from all independent in vitro experiments or from all individual mice from the indicated number of in vivo experiments. Statistical analyses were carried out by the Statistica software (StatSoft) using one-way ANOVA for evaluation of superoxide production and two-way (factorial) ANOVA for all other experiments. In case of kinetic assays, area under the curve was used for statistical analysis. All p values below 0.05 were considered statistically significant.
Results
In vitro differentiation of HoxB8 progenitors toward neutrophil-like cells
We have generated conditionally HoxB8-transduced progenitors by retroviral transduction of wild-type mouse bone marrow cells with an ER–HoxB8 fusion protein (18, 28). Progenitor cells were maintained in the presence of β-estradiol to maintain HoxB8 activity and conditioned media from SCF-producing CHO cells to promote progenitor cell survival and proliferation. The resulting cells (referred to as HoxB8 progenitors) are considered conditionally immortalized myeloid progenitor cells that can differentiate to neutrophils or other myeloid cells upon estrogen withdrawal and appropriate cytokine stimulation. As shown in the cytospin slides in Fig. 1A, such HoxB8 progenitors showed a typical morphology of early hematopoietic cells with large and uncondensed nuclei without features of final lineage commitment, with occasional cells apparently being in the stage of mitotic division.
Phenotypic characterization of in vitro–generated HoxB8 neutrophils. (A) Microscopic images of HoxB8 progenitor and HoxB8 neutrophil cell cultures. (B) Histograms of CD45, CD117, and Ly6G staining of HoxB8 progenitors and HoxB8 neutrophils. (C) Time course of CD117 and Ly6G expression level during neutrophilic differentiation of HoxB8 cells. (D) Time course of Ly6G positivity during neutrophilic differentiation of HoxB8 cells. (E) Histograms of various cell surface receptor staining of HoxB8 cells. Dead cells have been excluded from the analysis for (B)–(E). Microscopic images and flow cytometric histograms are representative of and quantitative data show mean and SEM from three to five independent experiments. Mean fluorescence intensity (MFI) normalized to the day 0 (CD117) or day 5 (Ly6G) value.
To induce neutrophilic differentiation of HoxB8 progenitors, the cells were cultured in the presence of SCF-containing supernatant and G-CSF without addition of β-estradiol. As shown in Fig. 1A, cells cultured under such conditions for 5 d (referred to as HoxB8 neutrophils) showed a morphology similar to that of mouse neutrophils with condensed and ring-shaped nuclei and less-prominent cytoplasmic staining. No dividing cells could be observed in such cultures (data not shown).
We have also performed flow cytometric analysis of HoxB8 progenitors and HoxB8 neutrophils, the latter ones after 5 d of culturing under conditions promoting neutrophilic differentiation. As shown in Fig. 1B, both HoxB8 progenitors and HoxB8 neutrophils expressed the general leukocyte marker CD45 at a similar level. In line with being early hematopoietic-lineage cells, HoxB8 progenitors also expressed the receptor tyrosine kinase CD117 (c-Kit), the receptor for the SCF cytokine. In line with their differentiation toward a single nonproliferative lineage, CD117 expression was completely lost in HoxB8 neutrophils. The expression of the neutrophil maturation marker Ly6G showed the opposite pattern: as expected from their noncommitted nature, Ly6G was absent from HoxB8 progenitors, whereas it was highly expressed on most HoxB8 neutrophils (Fig. 1B). It should nevertheless be noted that a proportion of HoxB8 neutrophils showed intermediate expression of Ly6G (Fig. 1B), and the peak Ly6G expression of in vitro–generated HoxB8 neutrophils was significantly lower than that of normal in vivo circulating neutrophils (data not shown), likely reflecting incomplete differentiation in cell culture.
We have also tested the time course of CD117 and Ly6G expression during neutrophilic differentiation of HoxB8 cells. As shown in Fig. 1C, the expression of CD117 dropped dramatically between days 1 and 3 of culture under conditions promoting neutrophilic differentiation and remained low until the end of the experiment (day 8). In contrast, Ly6G expression started to increase on day 3, reached its maximum on day 5, and remained around its maximum level until day 8.
Parallel to the analysis of average surface marker expression, we have also quantified the number of live cells in our cultures and divided them into Ly6G-negative and Ly6G-positive populations. As shown in Fig. 1D, the total number of cells moderately increased until days 4–5 with a dramatic increase of Ly6G-positive cells until day 5. Both the total number of cells and the number of Ly6G-positive cells started to decline from day 6, with a parallel increase of the number of dead cells (data not shown). This kinetic curve confirmed that day 5 is likely the most appropriate time for the analysis of HoxB8 neutrophils.
We have also tested expression of certain cell surface receptors involved in the functional activation of neutrophils (Fig. 1E). Expression of individual α-chains of major β2 integrins revealed that HoxB8 neutrophils and HoxB8 progenitors expressed similar levels of CD11a (integrin αL, the α-chain of LFA-1), whereas CD11b (integrin αM, the α-chain of Mac-1) showed robust upregulation during neutrophilic differentiation. Likely reflecting the differential changes of CD11a and CD11b expression, the levels of CD18 (the β-chain of all β2 integrins) showed a moderate increase in HoxB8 neutrophils compared with HoxB8 progenitors. We have also tested expression of FcγRs of myeloid cells. Although the expression of FcγRII/III was comparable between the HoxB8 progenitors and neutrophils (likely reflecting a broad expression in diverse myeloid lineages), FcγRIV showed strong upregulation in HoxB8 neutrophils. Again, a significant portion of HoxB8 neutrophils showed intermediate FcγRIV expression, likely reflecting incomplete differentiation in cell culture, especially because there was a strong overlap between the Ly6Gdim and FcγRIVdim cell populations (data not shown).
In vitro functions of HoxB8 neutrophils
We next tested the in vitro functional responsiveness of HoxB8 progenitors and HoxB8 neutrophils.
As shown in Fig. 2A, HoxB8 neutrophils mounted a robust respiratory burst upon stimulation with PMA, a nonphysiological stimulator of neutrophils, whereas no respiratory burst response from HoxB8 progenitors could be observed (p = 5.0 × 10−7). HoxB8 neutrophils, but not HoxB8 progenitors, also showed a robust respiratory burst response when plated on immobilized IgG immune complexes, a model of autoantibody-induced, Fc receptor–dependent neutrophil activation (39) (Fig. 2B; p = 5.5 × 10−5).
Functional responsiveness of in vitro–generated HoxB8 neutrophils. (A and B) Superoxide production of HoxB8 cells stimulated by PMA (A) or by plating on an immobilized IgG immune complex surface (B). Unstimulated control values were substracted. (C and D) Migration of HoxB8 cells toward various concentrations of fMLF (C) or MIP-2 (D) in a Transwell assay. (E and F) Phagocytosis of fluorescently labeled nonopsonized or mouse serum–opsonized S. aureus (E) or C. albicans (F) by HoxB8 cells. Mean and SEM of results from ten (A), nine (B), seven (C), and three (D) independent experiments are shown.
We also tested the chemotactic migration of HoxB8 progenitors and HoxB8 neutrophils through a fibrinogen-coated polycarbonate membrane of 5-μm pore size in an in vitro Transwell system. As shown in Fig. 2C, HoxB8 neutrophils were able to migrate toward an fMLF source with a maximum response at 1 μM fMLF, whereas no substantial migration of HoxB8 progenitors could be observed (p = 9.9 × 10−5). Similarly, HoxB8 neutrophils, but not HoxB8 progenitors, migrated toward the MIP-2 chemokine (p = 8.3 × 10−6) with a maximum response at 10 ng/ml MIP-2 (Fig. 2D).
Next, we tested phagocytosis of fluorescently labeled bacterial (S. aureus) and fungal (C. albicans) pathogens. As shown in Fig. 2E, no substantial phagocytosis of live GFP-expressing S. aureus (USA300 strain) was observed by HoxB8 progenitors, irrespective of whether the bacteria were opsonized by normal mouse serum or not. In contrast, HoxB8 neutrophils tended to show a moderate, although statistically NS, phagocytosis of nonopsonized S. aureus (Fig. 2E; p = 0.16 versus HoxB8 progenitors) and mounted a robust phagocytosis response of serum-opsonized bacteria (Fig. 2E; p = 1.3 × 10−9).
We have also tested phagocytosis of fluorescently labeled, heat-killed C. albicans yeast cells. As shown in Fig. 2F, HoxB8 progenitors failed to phagocytose the yeast cells, irrespective of prior opsonization. No significant phagocytosis of nonopsonized C. albicans by HoxB8 neutrophils could be observed either, whereas HoxB8 neutrophils showed robust phagocytosis of the serum-opsonized yeast cells (p = 2.7 × 10−4 versus HoxB8 progenitors) (Fig. 2F).
Taken together, our experiments presented so far indicate that HoxB8 neutrophils differentiated from HoxB8 progenitors in our hands are able to perform numerous in vitro functional responses typical of freshly isolated human or mouse neutrophils. This is in line with prior reports on the in vitro functions of these cells (28–30).
In vivo transplantation of HoxB8 progenitors
Having established an in vitro system for the generation of HoxB8 progenitor–derived neutrophils in our own hands, we next aimed to establish a system for the in vivo analysis of HoxB8 neutrophil function. To this end, we transferred HoxB8 cells into recipient mice. We assumed that the in vivo environment would provide more appropriate conditions for neutrophilic differentiation; therefore, we decided to transplant HoxB8 progenitors rather than in vitro–differentiated HoxB8 neutrophils. Because our HoxB8 cells expressed CD45.2 from their C57BL/6 genetic background, we chose CD45.1-expressing recipients to allow the identification of recipient- and donor-derived neutrophils by flow cytometry (38). The schematic design of the following experiments is shown in Fig. 3A.
Single transplantation of HoxB8 progenitors partially restores the neutrophil compartment. (A) Overview of the experimental design. (B) Comparison of donor- and recipient-derived circulating neutrophil counts in the different treatment groups. (C) Representative flow cytometric profiles of circulating neutrophils in HoxB8 chimeras at the indicated time points. (D) Percentage of donor- and recipient-derived neutrophils in the different tissues of HoxB8 chimeras on day 5. (E) Time course of donor- and recipient-derived circulating neutrophil counts of HoxB8 chimeras. Flow cytometric profiles are representative of and quantitative data show mean and SEM from 12–22 (B), 17–25 (C), 6 (D), and 17–25 (E) mice per group from 3 to 10 independent experiments, except for the nontransplanted, nonirradiated group (B), in which quantitative data are from two mice per group from two independent experiments.
We first transplanted HoxB8 progenitors i.v. into intact (nonirradiated) CD45.1-expressing recipients and compared donor- and recipient-derived circulating neutrophil counts with mice that did not receive HoxB8 progenitors. As shown in Fig. 3B, all mice contained only recipient-derived neutrophils on day 5, irrespective of whether they received HoxB8 progenitors on day 0. Those results suggested that HoxB8 progenitors are not able to efficiently engraft the recipients and differentiate into HoxB8 neutrophils in intact mice possibly because the hematopoietic niches in those recipients are fully occupied by recipient hematopoietic cells. Therefore, we next subjected recipient mice to lethal irradiation prior to the i.v. injection of HoxB8 progenitors. For comparison, we have also tested lethally irradiated mice that did not receive HoxB8 progenitors. As shown in Fig. 3B, the recipient neutrophils of lethally irradiated mice completely disappeared by day 5, irrespective of whether they received HoxB8 progenitors or not. This meant practically complete neutrophil deficiency in day 5 recipients that did not receive HoxB8 progenitors. In contrast, a clear donor-derived neutrophil population appeared in lethally irradiated recipients that received HoxB8 progenitors on day 0 (Fig. 3B), partially rescuing those mice from irradiation-induced neutropenia and replacing the neutrophil compartment with HoxB8 donor–derived cells.
We next aimed to further characterize the neutrophil compartment in lethally irradiated recipients transplanted with HoxB8 progenitors (referred to as HoxB8 chimeras). Fig. 3C shows the flow cytometric profile of such chimeras on day 0 (immediately before irradiation/transplantation) and on day 5, clearly showing that their circulating neutrophils (Ly6G-positive cells) consisted practically exclusively of recipient-derived (CD45.1) cells on day 0 and of donor-derived (CD45.2) cells on day 5. Quantitative assessment of a large number of such experiments indicated that on average, 97% of circulating neutrophils of HoxB8 chimeras on day 5 are of donor origin (Fig. 3D). Furthermore, analysis of tissue neutrophil compartments revealed that 80 and 83% of bone marrow and spleen neutrophils, respectively, were of donor origin 5 d after HoxB8 progenitor transplantation (Fig. 3D).
We have also tested the time course of the repopulation of the circulating neutrophil compartment by donor-derived cells. As shown in Fig. 3E, recipient-derived neutrophils practically completely disappeared by day 3, whereas donor-derived cells mainly appeared on day 5 when they also reached their maximum count at ∼40% of the initial circulating neutrophil count. Neutrophil numbers then began to decline, and the cells eventually completely disappeared by days 8–10. This was in line with the in vitro differentiation kinetics of HoxB8 cells (Fig. 1) and the expected inability of HoxB8 progenitors for long-term self-renewal without exogenous estrogen administration.
Besides transplanting pure HoxB8 progenitors into lethally irradiated recipients, we also tested two alternative approaches, resulting in the coexistence of neutrophils that have and have not undergone HoxB8 transduction and in vitro culture within the same individual chimeras. In the first competitive repopulation approach, lethally irradiated recipients were transplanted with untouched bone marrow cells, HoxB8 progenitors, or the mixture of these two cells (Supplemental Fig. 1A). As shown in Supplemental Fig. 1B and 1C, transplantation of bone marrow cells led to the gradual emergence of donor-derived neutrophils from approximately day 6 until the end of the experiment, whereas transplantation of HoxB8 progenitors resulted into a temporary increase of donor neutrophil counts that started to decline after a peak on day 6. Interestingly, addition of both donor cell types together in similar numbers as in Supplemental Fig. 1B and 1C resulted in the reduction of both bone marrow donor–derived and HoxB8 progenitor–derived neutrophil counts in the circulation (Supplemental Fig. 1D), suggesting that the two donor populations were competing for limited resources (e.g., niche factors) after repopulation. As a second alternative approach, we transplanted HoxB8 progenitors into sublethally irradiated recipients and followed the composition of the circulating neutrophil compartment (Supplemental Fig. 1E). As shown in Supplemental Fig. 1F, this resulted in the temporary coexistence of both donor-derived (HoxB8) and recipient neutrophils with a peak on day 6, whereas HoxB8 neutrophils disappeared from the circulation by day 10.
Phenotypic characterization of in vivo–differentiated HoxB8 neutrophils
We next aimed to perform a phenotypic characterization of in vivo–generated HoxB8 neutrophils. To this end, flow cytometry profiles of peripheral blood samples of HoxB8 chimeras on day 5 after HoxB8 progenitor transplantation were compared with those of intact C57BL/6 mice. Fig. 4A shows the forward-scatter (FSc) and side-scatter (SSc) profiles of all circulating leukocytes (stained using a pan-CD45 marker), with neutrophils (Ly6G-positive cells) highlighted in red. The figure indicates that in vivo–generated HoxB8 neutrophils have mostly normal size (FSc) and granularity (SSc), although their flow cytometric profile is a bit more spread out than in intact mice in both dimensions. In addition, the leukocyte populations characterized by low SSc (supposedly lymphocytes and monocytes) and by high SSc and high FSc (supposedly eosinophils) are less pronounced in HoxB8 chimeras than in intact mice. This finding likely reflects the fact that the lethal irradiation decimates all leukocyte populations, whereas HoxB8 progenitors primarily restore neutrophils with a limited ability to differentiate into other leukocytes.
Phenotypic characterization of in vivo–derived HoxB8 neutrophils. Flow cytometric profiles of peripheral blood leukocytes in intact mice and HoxB8 chimeras 5 d after irradiation. (A) FSc and SSc of all circulating leukocytes in a pan-CD45–positive gate. Neutrophils are marked in red. (B and C) Histogram of various cell surface receptor staining of neutrophils defined by their FSc and SSc characteristics (CD11a and FcγRII/III) or by Ly6G positivity (CD11b, CD18, and FcγRIV) within a CD45.2-positive gate. Representative data from two to five mice per group from three independent experiments are shown.
Fig. 4B and 4C show histograms of various cell surface markers of circulating neutrophils from intact C57BL/6 mice and day 5 HoxB8 chimeras. Practically all tested cells were positive for CD45.2 (data not shown), which was in line with the characteristic CD45.2-positive nature of intact C57BL/6 mice and the complete repopulation of the neutrophil compartment of HoxB8 chimeras by donor-derived cells (Fig. 3). Neutrophils from HoxB8 chimeras and intact mice showed a similar peak Ly6G level, suggesting nearly normal in vivo differentiation of HoxB8 progenitors toward neutrophils in vivo (Fig. 4B). Nevertheless, a minor population of Ly6Gdim neutrophils could consistently be found in HoxB8 chimeras (Fig. 4B). Importantly, however, this population was much less pronounced in circulating neutrophils of HoxB8 chimeras than in in vitro–differentiated HoxB8 neutrophils (compare Figs. 1B and 4B), suggesting a much more complete neutrophilic differentiation under in vivo conditions.
We have also tested expression of functionally important molecules on circulating neutrophils. As shown in Fig. 4C, the expression of the various β2 integrin chains (CD11a, CD11b, and CD18) and FcγRs (FcγRII/III and FcγRIV) were grossly similar on neutrophils from intact mice and HoxB8 chimeras. However, moderately increased expression of some of those molecules (including CD11b, FcγRII/III, and FcγRIV) could be observed in neutrophils of HoxB8 chimeras, possibly suggesting a somewhat primed state of the cells.
The increased expression of certain activating markers on HoxB8 neutrophils prompted us to test whether this was intrinsic to HoxB8-derived cells or was rather related to the irradiation/transplantation procedure. To this end, we tested expression of CD11b and FcγRIV on circulating neutrophils from intact and various chimeric mice. Neutrophils from chimeras generated using unfractionated bone marrow cells (bone-marrow chimeras) or HoxB8 progenitors (HoxB8 chimeras) showed a comparable increase in CD11b expression relative to cells from intact mice (Supplemental Fig. 2A–C). A similar observation could also be observed for FcγRIV expression (Supplemental Fig. 2D–F). We also took advantage of the coexistence of donor or recipient bone marrow–derived and HoxB8 progenitor–derived neutrophils in mixed bone marrow-chimeras (Supplemental Fig. 1D) and chimeras generated using sublethally irradiated recipients (Supplemental Fig. 1F). Analysis of the two neutrophil populations within the same individual mice revealed that there was no substantial difference between bone marrow–derived and HoxB8 progenitor–derived neutrophils in terms of CD11b (Supplemental Fig. 2G, 2H) or FcγRIV (Supplemental Fig. 2I, 2J) expression. Collectively, those results suggest that the increased CD11b and FcγRIV expression in HoxB8 chimeras is a consequence of the irradiation/transplantation procedure rather than an intrinsic feature of the HoxB8 system.
In vivo accumulation of HoxB8 neutrophils in thioglycollate-induced peritonitis
The above results indicated that transplantation of HoxB8 progenitors into lethally irradiated recipients resulted in the emergence of a substantial number of circulating HoxB8 progenitor–derived cells (HoxB8 neutrophils) that were phenotypically similar to intact mouse neutrophils. We next began testing the functional characteristics of those HoxB8 neutrophils. We first subjected HoxB8 chimeras to a thioglycollate-induced sterile peritonitis on day 5 after irradiation/transplantation. As shown in the flow cytometric profiles in Fig. 5A, thioglycollate triggered a robust infiltration of neutrophils into the inflamed peritoneum during the 4-h assay period. This infiltrating neutrophil population consisted practically entirely of donor-derived cells (Fig. 5B) in line with the practically complete absence of recipient-derived circulating neutrophils in these chimeras (Fig. 3). Further kinetic analysis (Fig. 5C) revealed that the majority of HoxB8 neutrophil infiltration occurred during the last 2 h of the assay period.
Peritoneal accumulation and phagocytic activity of in vivo–derived HoxB8 neutrophils. (A–C) Analysis of the peritoneal exudate upon thioglycollate-induced peritonitis. (A) FSc and SSc profiles of peritoneal cells in HoxB8 chimeras. (B and C) Quantitative analysis of donor- and recipient-derived peritoneal lavage neutrophils in HoxB8 chimeras 4 h (B) or the indicated times (C) after peritonitis induction. (D and E) Flow cytometric profile (D) or quantification (E) of phagocytosis of fluorescently labeled, heat-killed C. albicans by thioglycollate-elicited peritoneal neutrophils with or without 50 μg/kg latrunculin A treatment of HoxB8 chimeras. Dot plots are representative of and quantitative data show mean and SEM from 18 (A and B), 4–9 (C), or 6–12 (D and E) mice per group from six (A and B), five (C), and eight (D and E) independent experiments.
To estimate the efficacy of HoxB8 neutrophils in accumulating in the inflamed peritoneum, we compared the circulating and peritoneal numbers of neutrophils in intact mice and our HoxB8 chimeras. As shown in Supplemental Fig. 3A, the number of neutrophils in the peritoneum of HoxB8 chimeras during thioglycollate-induced peritonitis was ∼40% of that in intact mice, which was comparable to the ∼60% lower circulating neutrophil counts in HoxB8 chimeras (Fig. 3). In addition, analysis of all individual mice revealed a qualitatively similar relationship between circulating and peritoneal neutrophil counts in intact mice and HoxB8 chimeras (Supplemental Fig. 3B). Therefore, HoxB8 neutrophils appear to have a grossly similar capability of accumulating in the inflamed peritoneum as normal mouse neutrophils.
In vivo phagocytosis by HoxB8 neutrophils
We have also aimed to test the in vivo phagocytic capacity of HoxB8 neutrophils. To this end, thioglycollate-induced sterile peritonitis was triggered as above to induce extravasation of HoxB8 neutrophils, followed by the peritoneal injection of heat-killed, fluorescently labeled C. albicans 3 h after thioglycollate injection. After 1 additional h, the peritoneum was lavaged, and the phagocytosis of C. albicans by HoxB8 neutrophils was determined by flow cytometry. A parallel cohort of mice were also injected with latrunculin A, a known phagocytosis inhibitor, immediately before the injection of the heat-killed yeast cells. As shown in Fig. 5D and 5E, a substantial percentage of HoxB8 neutrophils phagocytosed the heat-killed C. albicans cells. This response was practically completely blocked by latrunculin A, confirming the role of the cytoskeletal machinery in the phagocytic response (p = 2.1 × 10−7).
Reverse passive Arthus reaction
The above experiments revealed that in vivo–generated HoxB8 neutrophils are able to perform various functional responses (chemotactic migration and phagocytosis) at a single-cell level. Our next question was whether the entire donor-derived neutrophil compartment as a whole is able to mediate neutrophil-dependent functional responses in HoxB8 chimeras. We chose the reverse passive Arthus reaction as a rapid, supposedly neutrophil-mediated (42) inflammatory response to test that question. To this end, HoxB8 chimeras were i.p. injected with OVA on day 5 after irradiation/transplantation, along with local intradermal injection of rabbit polyclonal anti-OVA Abs into one of the ears. Normal rabbit serum injection into the other ears of the same mice served as controls. Parallel experiments were also performed on irradiated, nontransplanted mice and, in some cases, intact C57BL/6 mice. The inflammatory reaction was tested by assessing the extravasation of the Evans blue dye and measurement of the ear thickness, as well as by determining the tissue accumulation of neutrophils and various inflammatory mediators.
As shown in Fig. 6A, edema formation indicated by blue staining of the ear (tissue accumulation of Evans blue) was triggered by anti-OVA, but not control treatment, in intact C57BL/6 mice. Similar changes could be seen in day 5 HoxB8 chimeras, but not in irradiated, nontransplanted mice (Fig. 6A). Measurement of the ear thickness (Fig. 6B) also confirmed anti-OVA–induced edema formation in intact mice and HoxB8 chimeras, but not in irradiated nontransplanted mice (p = 5.0 × 10−9 for comparison between the last two groups).
Reverse passive Arthus reaction in HoxB8 chimeras. Reverse passive Arthus reaction was triggered in intact mice; irradiated, nontransplanted mice; or HoxB8 chimeras by OVA, followed by intradermal injection of normal (Control) or anti-OVA rabbit serum into the indicated ears. (A and B) Inflammation and edema formation was tested by following the extravasation of i.v. injected Evans blue dye (A) or an ear thickness measurement (B). (C–E) Analysis of the accumulation of neutrophils (C), IL-1β (D), or MIP-2 (E) in ear samples. Images are representative of and quantitative data show mean and SEM from 6 to 10 mice per group from three independent experiments.
Next, we tested signs of inflammation in the ear tissue of HoxB8 chimeras and irradiated, nontransplanted control mice. As shown in Fig. 6C, a substantial neutrophil infiltration was observed in the anti-OVA–treated ears of HoxB8 chimeras, but not the contralateral control ears or in the ears of nontransplanted control animals (p = 3.1 × 10−4). Similarly, anti-OVA treatment triggered the accumulation of the proinflammatory cytokine IL-1β (p = 1.1 × 10−5) and the CXC chemokine MIP-2 (p = 0.043) in HoxB8-chimeras, but not in irradiated, nontransplanted mice (Fig. 6D, 6E).
Taken together, the above experiments indicate that HoxB8 chimeras are capable of undergoing complex inflammatory reactions. The fact that no such reactions could be observed in parallel-treated mice that were irradiated but did not receive HoxB8 progenitors indicates a critical role for HoxB8 progenitor–derived cells (supposedly neutrophils) in the inflammation process.
Longer-term maintenance of circulating HoxB8 neutrophils
After having tested the kinetics of repopulation of the neutrophil compartment and short-term, neutrophil-mediated inflammatory responses following a single injection of HoxB8 progenitors (Figs. 3–6), our next aim was to test whether it is possible to maintain a stable circulating HoxB8 neutrophil count for a longer period. To this end, we injected lethally irradiated recipients with HoxB8 progenitors in 2-d intervals (Fig. 7A). As shown in Fig. 7B, transplanting HoxB8 progenitors to lethally irradiated recipients immediately after irradiation as well as 2 and 4 d later resulted in the maintenance of a stable, circulating, donor-derived HoxB8 neutrophil population until 10 d after irradiation at ∼40% of the original circulating neutrophil count. Importantly, practically no recipient-derived neutrophils could be observed in these chimeras between 3 and 10 d after the irradiation and first HoxB8 progenitor injection.
Longer-term maintenance of HoxB8 neutrophils in vivo. Analysis of HoxB8 chimeras receiving repeated injections of HoxB8 progenitors. (A) Overview of the experimental design. (B) Quantitative analysis of peripheral blood neutrophil counts. Mean and SEM from 10 to 16 mice from two independent experiments are shown.
Autoantibody-induced arthritis in HoxB8 progenitor–transplanted recipients
The above experiments indicated that it is possible to maintain a mostly stable circulating HoxB8 neutrophil population by repeated injection of HoxB8 progenitors into lethally irradiated recipients. Therefore, our next aim was to test whether we can also induce longer-term inflammatory processes that are dependent on the function of the circulating donor-derived HoxB8 neutrophils. To this end, we turned to K/B×N serum–transfer arthritis, an autoantibody-induced in vivo model of autoimmune arthritis that is strongly dependent on neutrophil function (7, 43).
During the course of the next experiments (Fig. 8A), we have transplanted lethally irradiated CD45.1-expressing recipients with HoxB8 progenitors immediately after the irradiation (day 0) as well as every second days until day 10. The chimeras were also injected with arthritogenic (K/B×N) or nonarthritogenic control (B×N) serum on day 5. Their circulating neutrophil numbers (Fig. 8B) and disease course (Fig. 8C–E) were then followed for an additional 10 d (i.e., between days 5 and 15 from irradiation and first HoxB8 progenitor injection). We have also included intact C57BL/6 mice and irradiated, nontransplanted recipients in these experiments.
Autoantibody-induced arthritis in HoxB8 chimeras. K/B×N serum–transfer arthritis in intact mice; irradiated, nontransplanted mice; and HoxB8 chimeras. (A) Overview of the experimental design. (B) Quantitative analysis of peripheral blood neutrophil cell counts. (C–E) Analysis of arthritis development by photographing on day 7 (C), clinical scoring of the hind limbs (D), or ankle thickness measurement (E). Images are representative of and quantitative data show mean and SEM from three to six control and four to eight arthritic, serum-treated individual mice per group from three independent experiments.
Fig. 8B shows the time course of circulating neutrophil numbers in HoxB8 chimeras used in the above experiments. Similar to the experiments shown in Figs. 3D and 7B, recipient neutrophils practically completely disappeared by day 3 and remained so until the end of the experiment. Donor-derived neutrophils appeared in the circulation by day 5 (when arthritogenic and control serum was injected), and their numbers remained at around 40–50% of the initial neutrophil counts until the end of the experiment. No substantial difference in circulating neutrophil numbers could be observed between arthritogenic and control serum-treated HoxB8 chimeras (data not shown). Irradiated nontransplanted mice had practically no circulating neutrophils from day 3 after the irradiation until the end of the experiment (data not shown).
As shown in the photographs taken on day 7 after disease induction (Fig. 8C), arthritogenic, but not control serum injection, triggered robust arthritis in intact C57BL/6 mice. In contrast, no signs of arthritis could be observed in irradiated, nontransplanted mice. Importantly, however, injection of arthritogenic, but not control serum, also triggered robust arthritis in HoxB8 chimeras (Fig. 8C). Clinical scoring (Fig. 8D) and ankle thickness measurements (Fig. 8E) revealed that the disease course reached its maximum around day 7 after disease induction in arthritogenic serum-treated intact mice. Although arthritis development in irradiated, nontransplanted mice could only be tested for a shorter period of time (6 d after serum treatment [i.e., 11 d after irradiation]) because of their moribund state afterward, both the clinical scoring (Fig. 8D) and ankle thickness measurements (Fig. 8E) indicated complete lack of arthritis development in those animals. Importantly, arthritis development could also be observed in HoxB8 chimeras, which was similar to, or in the case of ankle thickening, even slightly more pronounced than that in intact mice (Fig. 8D, 8E). Statistical analysis indicated that arthritis development in HoxB8 chimeras was significantly different from that in irradiated, nontransplanted mice (p = 0.016 and 0.021 for clinical score and ankle thickness during days 0–6 from serum transfer, respectively) but it did not differ significantly from that in intact animals (p = 0.39 and 0.57, respectively).
These results indicate that prolonged maintenance of circulating neutrophil numbers by repeated injection of HoxB8 progenitors allows the development of highly complex long-term in vivo inflammation models, likely through the direct contribution from HoxB8 progenitor–derived neutrophils.
Other hematological parameters in HoxB8 chimeras
The aim of longer-term maintenance of circulating HoxB8 neutrophils and the moribund state of irradiated, nontransplanted mice prompted us to attempt a better understanding of the limitations of our approach. To this end, we tested various hematological parameters in irradiated, nontransplanted mice, as well as chimeras generated using a single transplantation of bone marrow cells or single or multiple transplantations of HoxB8 progenitors. All mice received a lethal irradiation on day 0. Hematological parameters were tested on days 0, 5, and 10, except for irradiated, nontransplanted mice that could not be tested on day 10 because of their moribund state. The lethal irradiation eliminated all recipient neutrophils, monocytes, B cells, and T cells (Supplemental Fig. 4A). Transplantation of bone marrow cells led to the emergence of donor-derived monocytes and B cells and, in this set of experiments, a smaller population of neutrophils, but not T cells (Supplemental Fig. 4A). Single or repeated transplantation of HoxB8 progenitors led to the expected transient and long-term emergence of donor neutrophils, respectively, whereas no other cell types emerged upon either HoxB8 progenitor transplantation approach (Supplemental Fig. 4A). We also tested other parameters, such as circulating RBC and platelet counts. As shown in Supplemental Fig. 4B, RBC counts were reduced to ∼30% of their original values in HoxB8 chimeras by day 10, whereas they practically returned to their normal range by the same day in chimeras receiving bone marrow donor cells. Similarly, HoxB8 chimeras had a practically complete absence of platelets by day 10, whereas a substantial number of circulating platelets could be observed in bone-marrow chimeras on the same day (Supplemental Fig. 4C). Taken together, lethal irradiation appears to eliminate all circulating leukocytes and platelets and to strongly reduce the number of circulating RBCs by day 10. Transplantation of bone marrow cells is able to revert most of those changes, whereas transplantation of HoxB8 progenitors reverts the loss of neutrophils, but not of other blood cell types. Nevertheless, this may be sufficient to maintain critical physiological functions, as indicated by the moribund state of irradiated, nontransplanted, but not HoxB8 progenitor–transplanted, mice.
Discussion
Neutrophils are critically involved in antimicrobial host defense during bacterial and fungal infections, as well as in various other diseases, including inflammatory diseases and cancer (1, 3, 4, 7). Unfortunately, understanding the functional biology of neutrophils is strongly hindered by their short-lived, terminally differentiated nature, limiting our options to genetically manipulate these cells. Indeed, proper genetic analysis of neutrophils has long been only possible by analyzing neutrophils from transgenic mice carrying germline mutations. A more recent approach, the conditional immortalization of myeloid progenitors by retrovirally expressed HoxB8 (generating so-called HoxB8 progenitors) allowed the in vitro culture and manipulation of cells that could later be differentiated to mature neutrophil-like cells (so-called HoxB8 neutrophils). Although this HoxB8-based approach is beginning to be widely used for in vitro studies, the in vivo potential of this system is still mostly unclear.
In this study, we worked out the conditions to replace the neutrophil compartment of recipient mice with HoxB8 neutrophils derived from in vitro–cultured HoxB8 progenitors. Using single or repeated injection of HoxB8 progenitors into lethally irradiated recipients allowed us to generate HoxB8 chimeras whose neutrophil compartment consisted practically exclusively of HoxB8 progenitor–derived cells. Those chimeras allowed us to perform single cell–based (migration and phagocytosis; Fig. 5) and whole population–based short-term (reverse passive Arthus reaction; Fig. 6) and long-term (K/B×N serum–transfer arthritis; Fig. 8) experiments, all of which were based on the functional capabilities of cells derived from previously in vitro–cultured HoxB8 progenitors. Those experiments extend the suitability of the HoxB8 system to detailed in vivo analysis of neutrophil function.
We decided to focus our work on the injection of HoxB8 progenitors, rather than in vitro–differentiated HoxB8 neutrophils, into the recipient mice. The reason for this was our feeling that the in vitro cultures provided suboptimal conditions for proper neutrophilic differentiation. This approach was retrospectively justified by the apparently more complete differentiation of the donor-derived neutrophils of HoxB8 chimeras than the in vitro–differentiated HoxB8 neutrophils (compare Figs. 1B and 4B). Another key point was to use lethally irradiated mice as recipients because HoxB8 progenitors were unable to engraft nonirradiated recipients (Fig. 3B). We hypothesize that HoxB8 progenitors need to occupy parts of the hematopoietic niches for their proper differentiation and the preoccupation of those niches by recipient-derived cells in nonirradiated recipients prevents the survival/differentiation of the donor progenitors. Our mixed bone marrow chimeric experiments also indicated competition between normal bone marrow–derived hematopoietic cells and HoxB8 progenitors (Supplemental Fig. 1A–D). In line with this, HoxB8 progenitors were unable to engraft nonirradiated Mcl1ΔMyelo recipients that have dramatic peripheral neutropenia (7) but a supposedly normal myeloid progenitor pool (A. Orosz and A. Mócsai, unpublished observations).
Flow cytometric analyses revealed interesting features of in vitro– and in vivo–generated HoxB8 neutrophils. One of the key findings was the consistently lower and inhomogeneous Ly6G expression in in vitro–generated HoxB8 neutrophils 5 d after estrogen removal (Fig. 1B) than in donor-derived neutrophils of HoxB8 chimeras on day 5 after HoxB8 progenitor transplantation (Fig. 4B). We assume that this is due to the suboptimal nature of the in vitro culture conditions for proper neutrophil development. Another consistent feature was that donor-derived neutrophils of HoxB8 chimeras on day 5 after HoxB8 progenitor transplantation showed upregulation of certain functional receptors (CD11b, FcγRII/III, and FcγRIV) compared with neutrophils of intact animals. This latter finding, however, is likely related to a proinflammatory, postirradiation environment (44) given that a similar upregulation could be observed upon transplantation of normal bone marrow cells into lethally irradiated recipients (Supplemental Fig. 2A–F) and that no substantial difference in CD11b and FcγRIV expression was seen between normal bone marrow–derived and HoxB8 progenitor–derived neutrophils within the same animals in mixed bone marrow chimeras and chimeras generated using sublethal irradiation (Supplemental Fig. 2G–J). The apparently primed state of circulating HoxB8 progenitor–derived neutrophils may also contribute to the normal or even enhanced inflammatory reaction of HoxB8 chimeras in the K/B×N serum–transfer model (Fig. 8C–E) despite the apparently reduced circulating neutrophil counts (40–50% of normal; Figs. 7B, 8B).
Although repeated transplantation of HoxB8 progenitors allowed HoxB8 chimeras to remain alive for 15 d after irradiation/first transplantation, we believe that it would be difficult to continue this procedure for a substantially longer time without negative effects on the overall health of the mice. We have noted that the mice were becoming weaker and less healthy toward the end of the 15-d period (data not shown). This is why we limited our K/B×N serum–transfer arthritis to 10 d rather than 14 d reported in our prior publications (7, 35, 36, 40, 41, 45, 46). In addition, irradiated, nontransplanted mice were moribund already by 10 d after transplantation. Our analysis of diverse hematopoietic lineages (Supplemental Fig. 4) suggests that irradiated, nontransplanted mice show multiple defects, including the absence of neutrophils, monocytes, and platelets, as well as a strong reduction of circulating RBC counts. In line with our original assumption, HoxB8 progenitors were only able to efficiently restore the neutrophil, but not other lineages, in vivo. This feature also provides an important limitation of our approach because the lack of other hematopoietic lineages hinders both the technical implementation and the later interpretation of experiments with HoxB8 chimeras. Nevertheless, the fact that HoxB8 chimeras remained apparently healthy for a prolonged period of time suggest that the wasting phenotype of irradiated, nontransplanted mice is significant in part because of the lack of circulating neutrophils.
Related to the issue of lineage specificity, it should be noted that there is no specific intervention in the generation or use of HoxB8 progenitors that would direct their differentiation toward the neutrophil lineage under in vivo conditions. We have nevertheless not observed substantial numbers of other donor-derived lineages such as monocytes, B or T cells, or eosinophils in our preliminary assessment (Supplemental Fig. 4 and data not shown). However, further detailed studies need to be performed to address this question in more detail.
It should also be noted that there have been prior attempts to analyze the in vivo function of HoxB8 neutrophils in recipient mice (20, 26, 31–33). Some of those studies used adoptive transfer of in vitro–generated HoxB8 neutrophils (26, 33), whereas others used cotransplantation of lethally irradiated recipients with a mixture of HoxB8 progenitors and normal bone marrow cells to maintain normal hematopoiesis (20, 31, 32). Therefore, both those approaches could only test single cell–based functional responses of HoxB8 neutrophils in the presence of recipient hematopoietic tissues, whereas our complete replacement approach allowed us to test functional (e.g., inflammatory) responses that required the entire neutrophil compartment. In addition, this is the first report, to our knowledge, that attempted the long-term maintenance of HoxB8 neutrophils, allowing the analysis of long-term functional responses of the cells. Finally, we also tried to perform a large number of control experiments (e.g., the inclusion of irradiated, nontransplanted controls mice) to provide the strongest possible evidence for the functional role of donor HoxB8 progenitor–derived neutrophils.
One of the main advantages of the HoxB8 system is that it allows genetic manipulation of myeloid progenitors that can later be differentiated toward neutrophils for functional analysis. This way, the generation and maintenance of genetically modified mice can be bypassed. We have already shown the suitability of this approach to test the role of the nonmuscle myosin H chain Myh9 in neutrophil migration in vitro (28). Our current study extends the suitability of the HoxB8 system to the in vivo analysis of neutrophil-mediated inflammation models in live mice.
Taken together, our experiments indicate that it is technically possible to establish and maintain circulating neutrophil populations derived from previously in vitro–cultured donor HoxB8 progenitors. This approach would strongly facilitate the analysis of the in vivo functional capacity of HoxB8 progenitor–derived neutrophils and allow the rapid in vivo testing of the effects of genetic modifications of HoxB8 progenitors on in vivo neutrophil function.
Disclosures
The authors have no financial conflicts of interest.
Acknowledgments
We thank Edina Simon and Nikolett Szénási for expert technical assistance; Annette Zehrer, Hanga Horváth, and Kata Szilveszter for help with the experiments; Krisztina Futosi for inspiring suggestions; Hans Häcker for the HoxB8-expressing retroviral vectors; Diane Mathis and Christophe Benoist for the KRN-transgenic animals; William Nauseef for the GFP-expressing S. aureus strain; Attila Gácser for the labeled C. albicans cells; and Anna Sebestyén for the preparation of cytospin slides.
Footnotes
This work was supported by the Hungarian National Research, Development and Innovation Office (Grants KKP129954 and NVKP_16-2016-1-0039 to A.M.), the Hungarian Ministry of National Economy (VEKOP-2.3.2-16-2016-00002 to A.M.), the Hungarian Higher Education Institutional Excellence Program, the Deutsche Forschungsgemeinschaft (SFB 914/A02 to B.W.), and the New National Excellence Program of The Hungarian Ministry for Innovation and Technology (ÚNKP-19-3-I-SE-42 to A.O.).
The online version of this article contains supplemental material.
Abbreviations used in this article:
- CHO
- Chinese hamster ovary
- FSc
- forward-scatter
- SCF
- stem cell factor
- SSc
- side-scatter.
- Received July 7, 2020.
- Accepted November 6, 2020.
- Copyright © 2021 by The American Association of Immunologists, Inc.
References
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