IL-17A induced granulopoiesis and megakaryopoiesis depends on the presence of TPO/c-mpl.
Keywords: inflammation, hematopoiesis, cytokines
Abstract
IL-17A is a critical, proinflammatory cytokine essential to host defense and is induced in response to microbial invasion. It stimulates granulopoiesis, leading to neutrophilia, neutrophil activation, and mobilization. TPO synergizes with other cytokines in stimulating and expanding hematopoietic progenitors, also leading to granulopoiesis and megakryopoiesis, and is required for thrombocytopoiesis. We investigated the effects of in vivo expression of IL-17A on granulopoiesis and megakaryopoisis in TPO receptor c-mpl−/− mice. IL-17A expression expanded megakaryocytes by 2.5-fold in normal mice but had no such effect in c-mpl−/− mice. The megakaryocyte expansion did not result in increased peripheral platelet counts. IL-17A expression did not impact bone marrow precursors in c-mpl−/− mice; however, it expanded splenic precursors, although to a lesser extent compared with normal controls (CFU-HPP). No peripheral neutrophil expansion was observed in c-mpl−/− mice. Moreover, in c-mpl−/− mice, release of IL-17A downstream cytokines was reduced significantly (KC, MIP-2, GM-CSF). The data suggest that IL-17A requires the presence of functional TPO/c-mpl to exert its effects on granulopoiesis and megakaryopoiesis. Furthermore, IL-17A and its downstream cytokines are important regulators and synergistic factors for the physiologic function of TPO/c-mpl on hematopoiesis.
Introduction
IL-17A is a known proinflammatory response cytokine, with no or only minimal expression under normal physiologic conditions [1]. It is induced primarily in activated T cells in response to infections with bacterial and fungal pathogens. IL-17A is critical for microbial host defense; in vivo expression profoundly stimulates hematopoiesis. In particular, it induces granulopoiesis via expansion of myeloid progenitors, leading to peripheral neutrophilia [2]. In clinical practice, thrombocytosis and related thrombotic events are often observed in patients with underlying inflammatory conditions, such as chronic infections, autoimmune diseases, or cancer [3]. Often, IL-17A has been reported to be overexpressed under these conditions [1, 4]. However, the exact basis for that phenomenon is not fully understood. The relationship between inflammation and thrombocytosis is intriguing, and it could be speculated whether there might be some involvement of the proinflammatory cytokine IL-17A.
One key regulator for megakaryopoiesis is TPO/c-mpl. c-mpl is an oncogene cloned from the genome of the murine myeloproliferative leukemia virus, which causes a myleoproliferative syndrome in mice. c-mpl was determined to be a hematopoietic cytokine receptor inducing thrombocytosis [5, 6]. Subsequently, its ligand TPO was cloned and confirmed to be a critical humoral factor that stimulates megakaryopoiesis and platelet formation [7, 8]. TPO/c-mpl regulates megakaryopoisis by driving progenitor cells into megakaryocyte proliferation and maturation. de Sauvage and coworkers [9] generated a viable c-mpl−/− mouse. Compared with normal littermates, its platelet counts were reduced substantially, although it did not suffer from spontaneous hemorrhages, and it had a normal lifespan. Although TPO/c-mpl is a dominant physiologic regulator in the process of platelet formation, this is still poorly understood in its entirety and likely depends on additional mechanisms, such as synergism with multiple other cytokines [10]. However, the actions of TPO/c-mpl are not only limited to megakaryopoisis, but TPO/c-mpl also plays a role in the expansion of early progenitors of other lineages, especially the myeloid lineage [11–13]. As c-mpl−/− mice have only reduced platelet counts but normal peripheral red and white cell counts, compensatory mechanisms and pathways for platelet production as well as granulopoiesis must exist.
We hypothesized TPO/c-mpl to synergize with IL-17A and its downstream cytokines in myeloid progenitor expansion and subsequent neutrophilia. We investigated the effects of in vivo IL-17A expression in c-mpl−/− mice on myelopoiesis and megakaryopoiesis. We investigated the effects of in vivo IL-17A expression in c-mpl−/− mice on myelopoiesis and megakaryopoiesis using a previously described adenoviral-based in vivo gene expression. Limitations of this system in hematopoiesis research have been reviewed previously [14].
MATERIALS AND METHODS
Animals
A generation of c-mpl−/− mice has been described previously and was provided by Dr. Fredric de Sauvage (Genentech, San Francisco, CA, USA) and coworkers [9]. Control mice matching their strain background (C57BL6) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). All animals were maintained under specific pathogen-free conditions in the animal facility of LSUHSC (New Orleans, LA, USA) and were 6–8 weeks of age at the time of the experiments. All experiments were conducted in accordance with LSUHSC Institutional Animal Care and Use Committee protocols. Construction, generation, expansion, and quality control of the AdmIL-17A and AdEGFP, as well as quality-control measures, have been described elsewhere [15, 16]. In preceding experiments, the optimal dose for AdmIL-17 was determined at 2.5 × 109 PFU and injected via the jugular vein. At this virus titer, no effects on readout parameters were observed using the control virus compared with saline-treated control animals. In vivo IL-17A expression using this technology was confirmed in animals and was consistent with expression data published previously [15].
Cell culture and cell isolation
Murine bone marrow was flushed from both femuri and filtered through nylon mesh for removal of tissue fragments. Spleens were ground between glass slides, and tissue fragments were filtered. Red blood cell lysis was accomplished by using hypotonic solution of NH4Cl, and mononuclear cells separated. Complete blood counts were performed on a Coulter counter (Beckman Coulter, Fullerton, CA, USA) or manually using peripheral blood smears.
Clonogenic progenitor assays (CFU-GM; -granulocyte, erythrocyte, monocyte, megakaryocyte; and -HPP), as well as their scoring, were performed using reagents obtained from (StemCell Technologies, Vancouver, BC, Canada; and Sigma, St. Louis, MO, USA). The procedures have been described elsewhere in detail [15]. The assays for quantification of megakaryocytic progenitors were performed using MegaCult-C kits purchased from StemCell Technologies. Assays were performed following instructions provided by the manufacturer (StemCell Technologies).
Cytokine analysis
Mouse plasma was analyzed for IL-1, IL-6, MIP-2, KC, and GM-CSF, using a multiplex assay (Bio-Rad, Hercules, CA, USA), according to the manufacturer's instructions. The plates were read using a Luminex 200 Total System machine (Luminex, Austin, TX, USA). The data were analyzed using LDS1.7 software. Murine TPO and IL-11 were analyzed using ELISA (Abcam, Cambridge, MA, USA).
Statistical analysis
Data were analyzed by ANOVA using the statistical program StatView (Abacus Concepts, Calabasas, CA, USA). Means of data were analyzed by ANOVA with Fisher's follow-up testing. A value of P < 0.05 was considered statistically significant.
RESULTS
In vivo IL-17A expression fails to expand peripheral neutrophils in c-mpl−/− mice
Seven mice/cohort (C57BL6 or c-mpl−/−) were injected with AdmIL-17A or AdEGFP. The ANC was determined using a Coulter counter; the results were verified by manual review of the smears. IL-17A failed to induce any significant increase in the peripheral neutrophil count at Day 7 in c-mpl−/− mice or any other time-point. In contrast, IL-17A induced a 3.5-fold neutrophil increase in normal C57BL6 mice (P<0.001; Fig. 1A). Neutrophil kinetics in control animals were consistent with data published previously showing the maximum at Day 7 [15].
Figure 1. In vivo IL-17A expression fails to expand peripheral neutrophils or platelet conts in c-mpl−/− mice.
Seven mice/strain (C57BL6 or c-mpl−/−) were injected with AdmIL-17A or AdEGFP. The ANC (A) and the platelet counts (B) were determined using a Coulter counter on Day 7; the results were verified by manual review of smears. Data represent the mean of seven animals ± se. These results were confirmed in two additional experiments. *P < 0.0005. MPL, c-mpl−/−.
In vivo IL-17A expression does not affect peripheral platelet counts in normal or c-mpl−/− mice
Seven mice/strain (C57BL6 or c-mpl−/−) were injected with AdmIL-17A or AdEGFP. IL-17A expression did not alter peripheral platelet counts in normal or c-mpl−/− mice. The platelet count was determined using a Coulter counter on Day 7; the results were verified by manual review of smears. No significant change in platelet counts were seen in either strain in response to IL-17A expression (Fig. 1B). Data represent the mean of seven animals ± se.
IL-17A fails to expands bone marrow CFU-GM and CFU-HPP in c-mpl−/− mice
Seven mice/strain (C57BL6 or c-mpl−/−) were injected with AdmIL-17A or AdEGFP. No effect was observed on frequency of bone marrow precursors (GM and HPP) when IL-17A was administered to c-mpl−/− mice. However, consistent with previous reported results, in normal control mice, a threefold increase (P<0.02) was observed for CFU-GM and a 5.6-fold increase for CFU-HPP (P<0.002; Fig. 2A and B) [15].
Figure 2. IL-17A expands spleen CFU-GM and -HPP in c-mpl−/− mice, however fails to expand bone marrow CFU-GM and CFU-HPP in c-mpl−/− mice.
Seven mice/strain (C57BL6 or c-mpl−/−) were injected with AdmIL-17A or AdEGFP. Spleens and bone marrow were harvested 7 days later. (A) Total bone marrow (BM) CFU-GM; (B) total CFU-HPP; (C) total spleen CFU-GM; (D) total spleen CFU-HPP. Results from each individual mouse were determined in quadruplicate. Data represent the mean of seven animals ± se. These results were confirmed in two additional experiments. *P < 0.005.
IL-17A expands spleen CFU-GM and -HPP in c-mpl−/− mice
Seven mice/strain (C57BL6 or c-mpl−/−) were injected with AdmIL-17A or AdEGFP. IL-17A expression increased splenic CFU-GM by 3.3-fold in normal C57BL6 mice and by fourfold in c-mpl−/− mice (P<0.001). IL-17A expression increased CFU-HPP by sevenfold in C57BL6 mice and 3.2-fold in c-mpl−/− mice (P<0.005; Fig. 2C and D).
IL-17A stimulates bone marrow-derived CFU-MKs in normal mice but not in c-mpl−/− mice
Seven mice/strain (C57BL6 or c-mpl−/−) were injected with AdmIL-17A or AdEGFP. Bone marrow CFU-MK were determined in quadruplicate for each mouse on Day 7. IL-17A expression more than doubled bone marrow CFU-MKs in C57BL6 mice (2.2-fold increase; P<0.01; Fig. 3). Although in c-mpl−/− mice, a slight increase was observed, it did not reach statistical significance.
Figure 3. IL-17A stimulates bone marrow-derived CFU-MKs in normal mice but not in c-mpl−/−.
Seven mice/strain (C57BL6 or c-mpl−/−) were injected with AdmIL-17A or control virus AdEGFP. Bone marrow CFU-MKs were determined in quadruplicate for each mouse on Day 7. Data represent the mean of seven animals ± se. These results were confirmed in two additional experiments. *P < 0.001.
IL-17 downstream neutrophil-stimulating cytokines are reduced in c-mpl−/− mice
Seven mice/strain and datapoint (C57BL6 or c-mpl−/−) were injected with Ad-mIL-17 or AdEGFP. MIP-2, KC, and GM-CSF serum cytokine levels were measured at defined time-points (0, 24, 48 h). Following IL-17A expression, MIP-2, KC, and GM-CSF levels were induced in normal animals (up to 3.5-fold) but only to a smaller degree (1.3-fold) for KC in c-mpl−/− mice (P<0.05; Fig. 4). MIP-2 and GM-CSF were not induced in c-mpl −/− animals. No changes were observed in plasma levels for IL-1, IL-6, and IL-11 in either mouse strain in response to IL-17 expression (data not shown).
Figure 4. IL-17A downstream neutophil-stimulating cytokines are reduced in cmpl−/− mice.
C57BL6 and c-Mpl−/− mice were injected with Ad-mIL-17 or AdEGFP (seven animals/strain, treatment, and time-point). Serum MIP-2, KC, and GM-CSF were determined at 24 and 48 h. Fold difference in plasma cytokine levels between animals treated with Ad-mIL-17 and AdEGFP is plotted at 24 and 48 h for (A) C57BL6 and (B) c-mpl−/− mice. These results were confirmed in two additional experiments. *P < 0.05.
DISCUSSION
Inflammation has long been known to be linked to stimulation of thrombocytopoiesis as well as to granulopoiesis. In previous work, despite IL-17A being a key proinflammatory cytokine, we did not observe or report statistically significant increases in peripheral platelet counts following IL-17A in vivo expression in normal mice, suggesting a lack of involvement in this process, at least for the final physiologically functional product [15, 17]. The question remained, however, whether there still might be some involvement in earlier steps of thrombocytopoiesis that might not be translated into elevated platelet counts.
In contrast, IL-17A and TPO are known stimulators of granulopoiesis. IL-17R−/− mice display normal baseline hematologic parameters, whereas c-mpl−/− mice have decreased peripheral platelets profoundly and also reduced progenitor cell pools. Upon microbial challenge, IL-17AR−/− animals fail to respond with appropriate emergency granulopoiesis and neutrophil stimulation [17, 18].
The data from knockout mice suggest that c-mpl−/− mice can compensate effectively for the lack of TPO/c-mpl and that c-mpl−/− mice, despite reduced progenitor pools, are still able to maintain near-normal granulopoiesis and maintain functional thrombocytopoiesis, albeit at reduced levels. Thus, IL-17R−/− and c-mpl−/− mice share similarities by having adequate compensatory mechanisms for their deficiencies that are at least sufficient to secure viability under normal baseline conditions. Although the compensatory mechanisms for either cytokine most likely involve different mechanisms, in light of their similar effects on granulopoiesis, we hypothesized some overlap or at least some interconnection between both. Another possibility could be that TPO might still have physiologic activity with other, thus far unknown, receptors. This was experimentally not addressed—only the absence of its presumed sole receptor cmpl. Previously, investigators hypothesized that certain cytokines might be able to compensate for the c-mpl−/−. Experiments with double-knockout mice demonstrated that G-CSF could compensate largely for the lack of TPO/c-mpl in granulopoiesis, but G-CSF had no effect on megakaryopiesis [19]. We investigated the hypothesis that IL-17 might stimulate thrombocytopoiesis via induction of other known cytokines (IL-11, leukemia inhibiting factor, and IL-6). However, expression levels remained unchanged for those in both normal and c-mpl−/− mice following IL-17 expression. We interpret these findings as lack of their involvement in the IL-17 induced megakayocyte expansion. [20].
Lineage development is controlled through cascades of complex cytokine networks. Given this complexity, it is likely that deficiency of only one critical cytokine at any step within this network would, at least, severely disturb the entire cascade. Many cytokines within a network require the presence of the other cytokines for synergistic activity; therefore, it is unlikely that simple overexpression of other cytokines within this network would restitute entirely normal physiology. Examples for altered and reduced bioactivity of cytokines in factor-deficient animals are, for instance, G-CSF or IL-17 expression in steel-Dickie mice [21]. This highlights the interdependence and the requirement for a physiologic cytokine milieu to achieve normal bioactivity of other cytokines in vivo.
It has been reported that TPO synergizes with G-CSF, Steel factor, and IL-6 in expanding and stimulating primitive progenitor cells as well as granulopoiesis [2, 22–24]. G-CSF, Steel factor (membrane-bound form), and IL-6 are also known downstream cytokines induced in vivo by IL-17A [1, 15, 21, 25]. Köhler et al. [26] recently reported G-CSF to trigger TPO induction in bone marrow of mice. In contrast to Köhler et al. [26], we did not detect TPO indiction in the plasma of mice treated with IL-17, although this does not rule out that such induction occurs at low levels within the hematopoietic microenvironment (data not shown). TPO, in turn, directly stimulates release of KC from megakaryocytes. At the same time, TPO requires KC as well as MIP-2 for neutrophil mobilization and mobility enhancement. MIP-2 and KC are also known, key IL-17A downstream cytokines [1, 17]. These interactions show how tightly IL-17A interconnects to the TPO/c-mpl mechanism via its downstream cytokines. Because of this relationship, it would be conceivable to postulate a physiologic relationship for both cytokines.
As established by others previously, our data reaffirm that TPO/c-mpl is not only a key regulator in thrombopoiesis but also of early granulopoiesis at the progenitor level [11]. IL-17A requires the presence of functional TPO/c-mpl to stimulate granulopoiesis effectively. We did not find IL-17A expression in c-mpl−/− mice to have any effect on bone marrow precursors. However, IL-17A still expanded splenic progenitors in c-mpl−/− mice, although CFU-HPP to a much lesser extent, compared with normal animals. Mature, spleen-derived colonies (CFU-GM) expanded similarly as those in normal control animals. This indicates a defect to IL-17A stimulation of more immature, hematopoietic elements in c-mpl−/− mice (bone marrow, CFU-HPP). One would expect expansion of granulopoietic CFU-GM to result in increased peripheral neutrophil counts. However, despite increased splenic CFU-GM, this was not seen in c-mpl−/− mice, and IL-17A expression was not sufficient to mobilize mature neutrophils into the periphery. We previously reported IL-17A to not only stimulate granulopoiesis but also to be a potent mobilizer for neutrophils and progenitor cells in vivo. This effect was explained by its release of G-CSF, MIP-2, KC, and stem cell factor [25]. As neither neutrophilia nor mobilization of peripheral progenitors (data not shown) was observed in c-mpl−/− mice after IL-17A administration, we conclude that TPO/c-mpl is required for the ability of IL-17A to expand myeloid progenitors and also to mobilize neutrophils and progenitor cells.
Although TPO synergizes with multiple other cytokines in progenitor expansion, it is not entirely clear as to why the release of IL-17A downstream mediators was reduced substantially in c-mpl−/− mice. However, these data would be consistent with the results reported by Köhler et al. [26], who demonstrated that TPO induced KC and MIP-2 production, although their observations were limited to release of these chemokines directly from megakaryocytes [26].
We found that IL-17A expanded bone marrow-derived CFU-MK in normal control mice but not in c-mpl−/− mice. One explanation would be that the effect of IL-17A and its downstream mediators on hematopoietic precursors depends on the presence and synergism with TPO [11]. Another hypothesis as to why c-mpl−/− megakaryopoiesis failed to respond to IL-17A could be that compensatory mechanisms in c-mpl−/− mice have already been exhausted or maximally stimulated, and therefore, no further response to IL-17A was possible. However, as we observed reduced release of secondary granulopoietic cytokines, it is more likely that c-mpl−/− mice are not responding appropriately to IL-17A and its downstream cytokines because of the lack of synergizing TPO/c-mpl.
Despite the increase in CFU-MK after IL-17A expression in normal mice, the peripheral platelet count did not change correspondingly as expected and remained unchanged. Platelet formation is a dynamic, multistep process that likely has different control mechanisms at each step of lineage development. The only conclusion that can be derived from our data is that although IL-17A expands megakaryocytes in normal mice, however, the final steps in platelet formation are independent of IL-17A or its downstream mediators. For that reason, IL-17A, by itself, likely would not explain elevated platelet counts in patients with underlying inflammatory diseases. Nevertheless, here, IL-17A may still be part of a derailed cytokine network that works in concert with additional inflammatory mediators leading to thrombocytosis. In summary, the data suggest that IL-17A requires the presence of TPO/c-mpl to stimulate megakaryopoiesis, and IL-17A cannot overcome the megakaryopoiesis defect in c-mpl−/−mice.
Although the data are consistent with the conclusion that TPO/c-mpl is required for IL-17A-mediated granulopoiesis and megakaryopoiesis, additional investigations will be needed to define further, additional details of these complex interactions.
Footnotes
- −/−
- deficient (knockout)
- AdEGFP
- control adenovirus-encoding EGFP
- AdmIL-17A
- adenovirus-encoding IL-17A
- ANC
- absolute neutrophil count
- c-mpl
- myeloproliferative leukemia virus oncogene encoding the thrombopoietin receptor
- CFU-HPP
- CFU of high-proliferating potential
- CFU-MK
- megakaryocyte CFU
- KC
- keratinocyte chemoattractant
- LSUHSC
- Louisiana State University Health Sciences Center
- TPO
- thrombopoietin
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