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Interferon-γ exerts dual functions on human erythropoiesis via interferon regulatory factor 1 signal pathway
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Interferon-g exerts dual functions on human erythropoiesis via interferon regulatory factor 1 signal pathway Wentian Wang a, 1, Huijuan Zhao a, b, 1, Yang Yang a, Ying Chi a, Xiang Lv c, **, Lei Zhang a, * a State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Tianjin Laboratory of Blood Disease Gene Therapy, CAMS Key Laboratory of Gene Therapy for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, 300020, China b Medical College, Henan University of Science and Technology, Luoyang, 471023, Henan, China c State Key Laboratory of Medical Molecular Biology, Department of Pathophysiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100005, China
a r t i c l e i n f o
a b s t r a c t s
Article history: Received 17 September 2019 Received in revised form 6 October 2019 Accepted 6 October 2019 Available online xxx
Hematopoiesis is systematically regulated by microenvironmental factors. The positive and negative factors coordinated together to yield a complicated blood system. Interferon-g (IFNg) has been identified as a common cause of various hematopoietic abnormalities, such as aplastic anemia. However, its impact on monolineage development, especially erythropoiesis, has not been fully elucidated from the cellular angle. In this study, we investigated the behavior of IFNg and found that IFNg plays dual functions on erythropoiesis; it not only blocks the erythroid lineage commitment but also accelerates the erythroid differentiation process, ultimately leading to the erythropoietic window clearance. IFNg can even powerfully initiate early differentiation without the existence of erythropoietin (EPO). Interferon regulatory factor 1 (IRF1) was confirmed as the essential downstream effector, and its ectopic overexpression can also have the same effect as that of IFNg. These results reveal that the IFNgeIRF1 axis plays a bidirectional role on erythropoiesis, impeding the access to erythroid lineage and driving the coming cells toward the differentiation endpoint. This model may place an innovative implication for IFNgeIRF1 axis to understand its in-depth mechanism on normal hematopoiesis and abnormal blood disorders, especially aplastic anemia. © 2019 Elsevier Inc. All rights reserved.
Keywords: Interferon-g Interferon regulatory factor 1 Erythropoiesis Differentiation process
1. Introduction The extracellular microenvironment is responsible for the destiny of hematopoietic stem cells (HSCs), leading to the vibrant and homeostatic hematopoiesis [1]. Erythropoiesis is the natural output as a result of hierarchical differentiation derived from HSCs [2], precisely controlled by a unitive and complicated network [3]. Various cell factors were found to participate in the regulatory network, the abnormality of which often causes physiological dysfunction. Besides certain positive cell factors that promote
* Corresponding author. Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College. ** Corresponding author. Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College. E-mail addresses: [email protected] (X. Lv), [email protected] (L. Zhang). 1 Equal contribution to this work.
erythropoiesis [4,5], several negative-regulating cytokines are also present in the microenvironment, including interferon-g (IFNg) [6]. Although the IFNg expression is nearly undetectable in healthy donors, its aberrantly high expression is seriously considered as a critical pathogeny of aplastic anemia (AA) [7]. In a mice model, long-term chronic IFNg accumulation induced by CD70 transgene intensely reduced erythroid output and hemoglobin content [8]. Similarly, parasitic infection could stressfully activate the IFNg expression [9], resulting in infectious anemia. Nevertheless, mycobacteria-infected mice produced extensive granulocytes and macrophages via extramedullary hematopoiesis and eventually died of severe infection while IFNg was knocked out, suggesting the indispensable role of IFNg in stressful hematopoiesis [10]. Although IFNg has been widely investigated in the blood system, revealing its negative impact on hematopoiesis and blood disorders [11], its direct mechanism on monolineage development, especially erythropoiesis, has not been well documented. It was confirmed that IFNg could strongly block erythroid commitment at
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Please cite this article as: W. Wang et al., Interferon-g exerts dual functions on human erythropoiesis via interferon regulatory factor 1 signal pathway, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.068
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the progenitor stage [12], but its influence on the differentiation process has not been fully elucidated. Thus, the cellular characteristic and molecular mechanism of IFNg in erythropoiesis still need in-depth analysis. In this study, we found that the IFNgeIRF1 axis could significantly accelerate erythroid differentiation, even without the existence of EPO. This study innovatively discovered a new insight on IFNg in hematopoiesis, placing valuable implications for erythroid-related diseases. 2. Materials and methods 2.1. Cell culture Human umbilical cord blood was collected from Tianjin Central Hospital of Gynecology Obstetrics, with the approval of the Ethics Committee of the Institute of Hematology & Blood Diseases Hospital (No. KT2015019-EC-1). The isolation and cultivation of CD34þ HSCs were performed in compliance with Miltenyi instructions and previous references [13,14], with soft optimization. For drug administration, 20 or 100 ng/mL IFNg was added into the liquid phase, from which the treated cells were obtained for further examination. Colony formation cell (CFC) assay was performed according to the manual of STEMCELL Technologies. A 293T cell line was cultured according to the standard protocol of ATCC. For lentivirus production, 293T cells were cotransfected by package plasmids according to Thermo and Clontech protocol with optimized modification. Further details are described in the supplementary materials. 2.2. Flow cytometry analyses For differentiation analysis, approximately 0.2 million cells were collected and incubated with antibodies for 30 min at room temperature. Regarding the cell cycle, cells were stained by BrdU and 7AAD according to the manufacturer’s protocol (BD). After two turns of regular washing, cells were examined by FACSCanto II, and the result was analyzed using the FlowJo software, version 10. 2.3. Real-time quantitative PCR (RT Q-PCR) The total RNA from cultured cells was extracted and reversely transcribed with oligo (dT)12VN using the PrimeScript kit according to the manufacturer’s protocol (Takara Bio). RT Q-PCR assay was conducted to define the expression abundance of concerned genes (Supplementary Table 1) using Thermo SYBR Green qPCR SuperMix-UDG with the ABI 7500 Real-Time PCR System. A melt curve analysis was additionally performed after amplification to verify the PCR product specificity. The expression pattern of all tested genes was calculated and normalized using GAPDH as the internal control by the 2-DCt or 2-DDCt algorithm. 2.4. RNAseq analysis A high-throughput RNAseq database was established in line with the previous reference [15]. The DNA library was successively constructed through a series of standard steps, with subsequent sequencing on the Illumina HiSeq platform (Novogene Co., Ltd., Beijing, China). Clean data was generated after filtering low-quality raw data. Transcriptional analysis, such as heatmap, GEO and GSEA, was conducted using the Novogene online tool. 2.5. Vector construction and gene disturbance IRF1 ORF was cloned into the lentivirus vector, fused with sfGFP ORF at the C-terminus and followed by the downstream puromycin
resistance gene. For transduction, 0.5 million/mL HSCs were incubated with lentivirus and polybrene (8 mg/mL) for 12e24 h. The infected cell population was selected by 0.5 mg/mL puromycin, as shown in Fig. 4A. 2.6. Statistical analysis All data were analyzed and presented as mean ± SD. Significant differences between the two groups were determined using a twotailed Student’s t-test using the GraphPad Prism software, version 8.02. A P-value of <0.05 indicated a statistically significant difference. 3. Results 3.1. IFNg significantly accelerated the erythroid differentiation process To evaluate the potential function of the IFNg signals, a classic in vitro liquid culture system was used to mimic the natural erythropoiesis process (Fig. 1A). As a result, HBA1/2 globin genes and EPOR were highly activated (Fig. 1B and C), verifying the reliable culture system for erythropoiesis. The IFNg receptors, IFNGR1 (Fig. 1D) and IFNGR2 (Fig. 1E), had slight changes, maintaining relatively stable activity during differentiation, suggesting the potential openness of IFNg in erythropoiesis. Subsequently, the erythroid differentiation process in IFNg administration was continuously monitored. Primarily, cell proliferation increased in the first 2 days of culture (Fig. 1F, Supplementary Fig. 1), in line with the results of a study [16]. However, cell mitosis was suppressed in long-term treatment on day 8 (Fig. 1G), in line with another result [8]. Despite the paradoxical influence on cell proliferation, the differentiation process was remarkably accelerated by IFNg (Fig. 1H). One representative experiment was conducted, as shown in Fig. 1I, exhibiting the substantial cell population shift from CD235- to CD235þ. Besides the differentiation process was accelerated at the cellular level, the action was also hastened at the gene expression level (Supplementary Fig. 2). The erythroid-specific genes HBA1/2 were undoubtedly activated (Fig. 1J), while the transcriptional factor GATA1 was mildly promoted (Fig. 1K), implying some additional mechanism partly independent of the classic erythroid network was involved in the differentiation acceleration. Finally, the blocking effect of IFNg on commitment was also evaluated, revealing that the three commitments were all inhibited, especially BFU-E cells (Fig. 1L). 3.2. IFNg forcefully initiated early differentiation via an EPOindependent pathway IFNg potently pushed cells into the CD71þCD235þ population, accelerating the differentiation process toward erythrocytes. However, it was unclear whether this drive will still occur or stall if the crucial power EPO was withdrawn. To verify this, a similar culture system was used, with or without IFNg application (Fig. 2A). Unexpectedly, IFNg can still forcefully initiate early differentiation even without the existence of EPO. The CD71þCD235- and CD71þCD235þ population in IFNg administration was strikingly enhanced on day 4 (Fig. 2B and C). Besides 100 ng/mL IFNg, a lower concentration was also applied. The representative FACS results were shown in Fig. 2D, from which the 20 ng/mL group obtained a similar but slightly softer result. Furthermore, critical receptors for erythropoiesis were examined. Interestingly, c-KIT was downregulated (Fig. 2E), while EPOR was upregulated (Fig. 2F), indicating the opened differentiation access. Moreover, HBA1/2 and GATA1 were both upregulated (Supplementary Fig. 3A and B). All the
Please cite this article as: W. Wang et al., Interferon-g exerts dual functions on human erythropoiesis via interferon regulatory factor 1 signal pathway, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.068
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Fig. 1. IFNg significantly accelerated the in vitro erythroid differentiation process. (A) Flow chart displayed the in vitro culture program for erythropoiesis. (B-C) HBA1/2 globin (B) and EPOR (C) genes were monitored to confirm successful differentiation. Cells at checkpoints were collected and disposed of for RT Q-PCR. Statistical differences were analyzed from biological repeats. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (D-E) Relative expression abundance of IFNg receptors, IFNGR1 (D) and IFNGR2 (E), was evaluated. (F-G) Cell proliferation was calculated in short-term (F) and long-term (G) culture with 100 ng/mL IFNg treatment. (H) The CD71þCD235þ population on day 6 was statistically analyzed. FACS was performed by labeling cells with indicated antibodies. (I) The FACS results of one representative experiment was shown. 100 ng/mL IFNg was added in the liquid phase, and cells were collected and subsequently labeled with antibodies as indicated at each checkpoint. (J-K) RT Q-PCR was performed to monitor erythroid gene expression (J, HBA1/2; K, GATA1) under IFNg exertion on day 6. (L) CFC assay was performed to confirm the reported effect of IFNg on lineage commitment. After the 6-day pre-expansion, a total of 1000 cells were seeded into H4434 methylcellulose semi-solid medium (containing SCF, IL-3, EPO, and GM-CSF). Meanwhile, 100 ng/ml IFNg or PBS were added into the medium for the 14-day CFC formation. Then the mature colonies were characterized via microscopic examination.
abovementioned results suggested that IFNg was responsible for the stressful initiation of early erythropoiesis via an EPOindependent mechanism.
3.3. The transcription profile of hematopoietic cells was remodeled by IFNg administration Results had revealed a novel function of IFNg on erythropoiesis,
even strongly priming the early differentiation independent of EPO. However, the action of IFNg and involved mechanisms were still unclear. To determine these, a high-throughput RNAseq assay was performed. Four groups were involved, and the total transcription profile was presented as a heatmap (Fig. 3A). The two IFNg-incubated groups showed a resembling expression pattern, significantly distinct from the untreated ones. Considering the equal influence of IFNg on days 2 and 4 (Fig. 2D), it was reasonable that the common-
Please cite this article as: W. Wang et al., Interferon-g exerts dual functions on human erythropoiesis via interferon regulatory factor 1 signal pathway, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.068
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Fig. 2. IFNg initiated early erythroid differentiation independent of EPO. (A) The flow chart displayed the pre-differentiation culture system without EPO. (B-C) FACS results on day 4 were statistically analyzed, including the CD71þCD235- population (B) and CD71þCD235þ population (C). (D) One representative experiment was presented, including the day 2 and 4 results in 20 or 100 ng/mL IFNg administration. (E-F) RT Q-PCR determined the expression change of c-KIT (E) and EPOR (F) on day 4 with 100 ng/mL IFNg application.
regulated 453 genes were assumed as the potential pool for mechanism excavation (Fig. 3B). Next, GEO classifications and GSEA analyses were performed. The interferon signal pathway was profoundly enriched in the IFNg day 4 sample (Fig. 3C), similar to the GSEA results (Supplementary Fig. 3C). The heme metabolism was especially enriched (Supplementary Fig. 3D), implying the potential role of IFNg on hemoglobin production. Notably, the top upregulated genes were presented, among which IRF1 was noted (Fig. 3D, Supplementary Table 2). Moreover, the IRF family, IRF1 to IRF9, were further determined by RT Q-PCR, revealing that IRF1 was indeed the most motivated gene and had the highest abundance (Fig. 3E), suggesting IRF1 as the most reasonable effector for IFNg signal.
the gene transcription was also modulated. Both HBA1/2 (Fig. 4F) and GATA1 (Fig. 4G) were upregulated in IRF1 administration. Considering the slight change in SPI1 expression with IFNg administration (RNAseq data), it was reasonable that the IFNgeIRF1 axis might function through a pathway partly without SPI1. Due to the abnormal activation of IFNg in certain blood disorders, such as AA [6,11,17], its effector IRF1 also needs to be determined in related patients. The results revealed that IRF1 was activated in patients with AA (Fig. 4H), compared to healthy donors and those with myelodysplastic syndrome. All the abovementioned data proposed a novel hypothesis that the IFNgeIRF1 axis negatively blocked BFUE at the commitment stage and significantly accelerated the differentiation process at the later stage, leading to cleared erythropoietic windows and ultimately causing anemia (Fig. 4I).
3.4. IRF1 overexpression played dual functions on erythropoiesis just like IFNg
4. Discussion
To assess the latent value of IRF1, the overexpression of IRF1 was achieved using an optimized lentiviral vector (Supplementary Fig. 4). The infected primary cells were put into puromycin selection and EPO-driven differentiation, or CFC-assay as shown in Fig. 4A. The IRF1 enhanced group formed smaller and fewer colonies while the BFU-E colonies were more affected (Fig. 4B), indicating the blocking role of IRF1 on commitment. Subsequently, IRF1 performance on the erythroid process was further analyzed. First, IRF1 enforcement was confirmed by RT Q-PCR (Fig. 4C). Then, the erythropoietic process was continuously monitored. Surprisingly, ectopic IRF1 overexpression led to differentiation acceleration (Fig. 4D). The statistical analysis showed that IRF1 could powerfully and reproducibly augment the CD71þCD235þ population (Fig. 4E). Moreover, not only was the cell characteristic remodeled by IRF1,
A few studies have explored the impact of IFNg on erythropoiesis. It was of interest that the early-stage CD71þ population increased in mice under chronic IFNg accumulation, but with a lower output of erythrocytes [8,18]. In fact, it was reasonable to recognize this phenomenon as a compensatory consequence of the increased EPO, raising corrective feedback to anemia. In this study, a more accurate strategy was performed in vitro, and a systematic conclusion was deduced. To our knowledge, it was the first time that IFNg was found to accelerate the differentiation process as a dual regulator, leading to erythropoietic window clearance, and the dual influence of IRF1 described here was also unreported previously. Interestingly, except IFNg, TGFb was also found to play comparatively similar functions, not only reducing BFU-E proliferation but also accelerating downstream differentiation [19],
Please cite this article as: W. Wang et al., Interferon-g exerts dual functions on human erythropoiesis via interferon regulatory factor 1 signal pathway, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.068
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Fig. 3. Bioinformatical analysis excavated the molecular mechanism of IFNg in differentiation acceleration. (A) The RNAseq transcription profile of the 4 groups was shown in the heatmap. The log10 (FPKMþ1) values were normalized and clustered. The red color indicated high expression, while the blue color indicated low expression. (B) The Wayne diagram showed the overlap of different genes among different comparison combinations. The numbers in the circle represent the sum of both upregulated (2-fold) and downregulated (0.5-fold) genes in the comparative combinations. (C) As a result of GO enrichment analysis, the most significant 20 terms were selected to draw a histogram. It can determine which biological functions or pathways were significantly enriched. (D) The top 8 upregulated trans factors in IFNg administration were arranged in table. (E) The IRF family was systematically scanned by RT Q-PCR to select the exact member for IFNg signal. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
indicating the resembling functions of these negative cell factors on hematopoiesis. The cytokine receptors, signal adaptors, and trans factors were all considered in the mechanism. Instead of c-KIT and EPOR being suppressed as previously reported [20], EPOR was obviously activated in our study, consistent with the differentiation acceleration. Moreover, a similar difference in GATA1 between another study [21] and our work was also noted. This contradiction may be partially explained by time, because cells may adjust their response to IFNg stimulation with time. For instance, cell proliferation was efficiently enhanced in short time culture, coincident with some results [16,22], but strongly impaired in long term culture, similar to other findings [8]. Thus, the temporary stress and long term influence would switch diversely, suggesting the time-axis significance of IFNg in erythropoiesis. It was confirmed that several vital signals were involved in IFNg-administrated erythropoiesis. STAT1, the critical effector of IFNg signal, and JAK2, the essential adaptor of EPO signal, were both dramatically stimulated (Supplementary Table 2), similar to the protein modification previously described [17]. Stat1 favored megakaryopoiesis and inhibited erythropoiesis [23,24], which slightly contradicts the result of this study, but it was also reported that EPO could similarly activate Stat1 as downstream signal [25],
implying the complicated role of Stat1 in erythropoiesis. Interestingly, Stat1-deficient erythroid cells revealed a less differentiated phenotype [26], implying its potential regulatory role on erythropoiesis, consistent with the upregulated STAT1 expression along the accelerated differentiation. However, the upregulation of STAT1 may be partially due to its self-control feedback [27]. It was also unlikely that IFNg mainly functioned through the JAK2 pathway because all downstream erythroid-specific trans factors were only softly modulated or had slight changes. In this study, we instead paid more attention to resurvey the remodeling of transcriptional network. Based on high-throughput RNAseq data, IRF1, a crucial immunological protein against viral and bacterial infection [8,28], was identified. Unlike earlier findings that IRF1 controlled granulopoiesis instead of erythropoiesis [29] or coordinated with SPI1 as the inhibitor of erythropoiesis [8], it was revealed in our survey as an energetic speeder for erythroid differentiation, similar to IFNg. Recently, the IFNgeIRF1 axis is still concerned in hematopoiesis and blood disorders [6,11,16,30e33]. Some abnormal IFNg expression was even detected in the plasma of patients with polycythemia vera, implying the likely feedback of IFNg on increased erythropoiesis [34]. Our findings may outline a beneficial principle for the IFNgeIRF1 axis on hematopoiesis and endow IFNg new potential value for blood disorder therapy.
Please cite this article as: W. Wang et al., Interferon-g exerts dual functions on human erythropoiesis via interferon regulatory factor 1 signal pathway, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.068
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Fig. 4. Ectopic IRF1 overexpression played dual functions in erythropoiesis. (A) The vector construction (upper) and culture flow (lower) were presented. The lentiviral vector carrying sfGFP alone without IRF1 was used as a control vector, and both groups were selected under 0.5 mg/mL puromycin. (B) The colony assay under IRF1 overexpression was performed. After transducted for 3 days, approximately 500 transduced primary cells were seeded in H4434 medium and meanwhile supplied with 0.1 mg/mL puromycin. After 14day culture, the formatted colonies were counted. (C) RT Q-PCR was performed to determine ectopic IRF1 expression in primary cells. Cells on day 2 were collected and disposed of for RT Q-PCR. (D) The cellular differentiation process was monitored, and representative results were arranged. Cells were collected, stained and detected by FACS. (E) Biological repeats were also executed to gain statistical results. (F-G) The expression pattern of HBA1/2 (F) and GATA1 (G) were examined by RT Q-PCR. Cultured cells on day 5 were gathered for RT Q-PCR. (H) IRF1 expression was assessed in peripheral blood mononuclear cells (PBMCs) from normal healthy donors and patients. The PBMCs were separated by Ficoll density centrifugation, with subsequent RT Q-PCR. (I) A novel IFNgeIRF1 function model was hypothesized. IFNgeIRF1 axis may play two disparate effects on erythropoiesis, blocking erythroid lineage commitment and accelerating erythroid differentiation process, leading to erythropoietic window clearance.
Therefore, this study systematically explored the potential function of IFNg in erythropoiesis and proposed a novel hypothesis to demonstrate the dual functions of the IFNgeIRF1 axis in erythropoiesis, which may place valuable implications for understanding and treatment of erythroid-related diseases. Disclosure of conflicts of interest
(19JCZDJC33000, 18JCQNJC11900); The Beijing-Tianjin-Hebei basic research project (18JCZDJC44600/H2018206423); Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (2017PT31047, 2018PT31038, 2018PT32028); CAMS Innovation Fund for Medical Sciences (2016-I2M-1-018, 2017-I2M1-015); Novo Nordisk Haemophilia Research Fund in China, Denmark. We thank Yahong You for supplying several blood samples from patients.
All the authors declare no competing financial interests. Acknowledgements Appendix A. Supplementary data This work was supported by National Natural Science Foundation of China (81500084, 81970121, 81600099, 81900126); Tianjin Municipal Science and Technology Commission Grant
Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.10.068.
Please cite this article as: W. Wang et al., Interferon-g exerts dual functions on human erythropoiesis via interferon regulatory factor 1 signal pathway, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.068
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Interferon-g exerts dual functions on human erythropoiesis via interferon regulatory factor 1 signal pathway Wentian Wang a, 1, Huijuan Zhao a, b, 1, Yang Yang a, Ying Chi a, Xiang Lv c, **, Lei Zhang a, * a State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Tianjin Laboratory of Blood Disease Gene Therapy, CAMS Key Laboratory of Gene Therapy for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, 300020, China b Medical College, Henan University of Science and Technology, Luoyang, 471023, Henan, China c State Key Laboratory of Medical Molecular Biology, Department of Pathophysiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100005, China
a r t i c l e i n f o
a b s t r a c t s
Article history: Received 17 September 2019 Received in revised form 6 October 2019 Accepted 6 October 2019 Available online xxx
Hematopoiesis is systematically regulated by microenvironmental factors. The positive and negative factors coordinated together to yield a complicated blood system. Interferon-g (IFNg) has been identified as a common cause of various hematopoietic abnormalities, such as aplastic anemia. However, its impact on monolineage development, especially erythropoiesis, has not been fully elucidated from the cellular angle. In this study, we investigated the behavior of IFNg and found that IFNg plays dual functions on erythropoiesis; it not only blocks the erythroid lineage commitment but also accelerates the erythroid differentiation process, ultimately leading to the erythropoietic window clearance. IFNg can even powerfully initiate early differentiation without the existence of erythropoietin (EPO). Interferon regulatory factor 1 (IRF1) was confirmed as the essential downstream effector, and its ectopic overexpression can also have the same effect as that of IFNg. These results reveal that the IFNgeIRF1 axis plays a bidirectional role on erythropoiesis, impeding the access to erythroid lineage and driving the coming cells toward the differentiation endpoint. This model may place an innovative implication for IFNgeIRF1 axis to understand its in-depth mechanism on normal hematopoiesis and abnormal blood disorders, especially aplastic anemia. © 2019 Elsevier Inc. All rights reserved.
Keywords: Interferon-g Interferon regulatory factor 1 Erythropoiesis Differentiation process
1. Introduction The extracellular microenvironment is responsible for the destiny of hematopoietic stem cells (HSCs), leading to the vibrant and homeostatic hematopoiesis [1]. Erythropoiesis is the natural output as a result of hierarchical differentiation derived from HSCs [2], precisely controlled by a unitive and complicated network [3]. Various cell factors were found to participate in the regulatory network, the abnormality of which often causes physiological dysfunction. Besides certain positive cell factors that promote
* Corresponding author. Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College. ** Corresponding author. Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College. E-mail addresses: [email protected] (X. Lv), [email protected] (L. Zhang). 1 Equal contribution to this work.
erythropoiesis [4,5], several negative-regulating cytokines are also present in the microenvironment, including interferon-g (IFNg) [6]. Although the IFNg expression is nearly undetectable in healthy donors, its aberrantly high expression is seriously considered as a critical pathogeny of aplastic anemia (AA) [7]. In a mice model, long-term chronic IFNg accumulation induced by CD70 transgene intensely reduced erythroid output and hemoglobin content [8]. Similarly, parasitic infection could stressfully activate the IFNg expression [9], resulting in infectious anemia. Nevertheless, mycobacteria-infected mice produced extensive granulocytes and macrophages via extramedullary hematopoiesis and eventually died of severe infection while IFNg was knocked out, suggesting the indispensable role of IFNg in stressful hematopoiesis [10]. Although IFNg has been widely investigated in the blood system, revealing its negative impact on hematopoiesis and blood disorders [11], its direct mechanism on monolineage development, especially erythropoiesis, has not been well documented. It was confirmed that IFNg could strongly block erythroid commitment at
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Please cite this article as: W. Wang et al., Interferon-g exerts dual functions on human erythropoiesis via interferon regulatory factor 1 signal pathway, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.068
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the progenitor stage [12], but its influence on the differentiation process has not been fully elucidated. Thus, the cellular characteristic and molecular mechanism of IFNg in erythropoiesis still need in-depth analysis. In this study, we found that the IFNgeIRF1 axis could significantly accelerate erythroid differentiation, even without the existence of EPO. This study innovatively discovered a new insight on IFNg in hematopoiesis, placing valuable implications for erythroid-related diseases. 2. Materials and methods 2.1. Cell culture Human umbilical cord blood was collected from Tianjin Central Hospital of Gynecology Obstetrics, with the approval of the Ethics Committee of the Institute of Hematology & Blood Diseases Hospital (No. KT2015019-EC-1). The isolation and cultivation of CD34þ HSCs were performed in compliance with Miltenyi instructions and previous references [13,14], with soft optimization. For drug administration, 20 or 100 ng/mL IFNg was added into the liquid phase, from which the treated cells were obtained for further examination. Colony formation cell (CFC) assay was performed according to the manual of STEMCELL Technologies. A 293T cell line was cultured according to the standard protocol of ATCC. For lentivirus production, 293T cells were cotransfected by package plasmids according to Thermo and Clontech protocol with optimized modification. Further details are described in the supplementary materials. 2.2. Flow cytometry analyses For differentiation analysis, approximately 0.2 million cells were collected and incubated with antibodies for 30 min at room temperature. Regarding the cell cycle, cells were stained by BrdU and 7AAD according to the manufacturer’s protocol (BD). After two turns of regular washing, cells were examined by FACSCanto II, and the result was analyzed using the FlowJo software, version 10. 2.3. Real-time quantitative PCR (RT Q-PCR) The total RNA from cultured cells was extracted and reversely transcribed with oligo (dT)12VN using the PrimeScript kit according to the manufacturer’s protocol (Takara Bio). RT Q-PCR assay was conducted to define the expression abundance of concerned genes (Supplementary Table 1) using Thermo SYBR Green qPCR SuperMix-UDG with the ABI 7500 Real-Time PCR System. A melt curve analysis was additionally performed after amplification to verify the PCR product specificity. The expression pattern of all tested genes was calculated and normalized using GAPDH as the internal control by the 2-DCt or 2-DDCt algorithm. 2.4. RNAseq analysis A high-throughput RNAseq database was established in line with the previous reference [15]. The DNA library was successively constructed through a series of standard steps, with subsequent sequencing on the Illumina HiSeq platform (Novogene Co., Ltd., Beijing, China). Clean data was generated after filtering low-quality raw data. Transcriptional analysis, such as heatmap, GEO and GSEA, was conducted using the Novogene online tool. 2.5. Vector construction and gene disturbance IRF1 ORF was cloned into the lentivirus vector, fused with sfGFP ORF at the C-terminus and followed by the downstream puromycin
resistance gene. For transduction, 0.5 million/mL HSCs were incubated with lentivirus and polybrene (8 mg/mL) for 12e24 h. The infected cell population was selected by 0.5 mg/mL puromycin, as shown in Fig. 4A. 2.6. Statistical analysis All data were analyzed and presented as mean ± SD. Significant differences between the two groups were determined using a twotailed Student’s t-test using the GraphPad Prism software, version 8.02. A P-value of <0.05 indicated a statistically significant difference. 3. Results 3.1. IFNg significantly accelerated the erythroid differentiation process To evaluate the potential function of the IFNg signals, a classic in vitro liquid culture system was used to mimic the natural erythropoiesis process (Fig. 1A). As a result, HBA1/2 globin genes and EPOR were highly activated (Fig. 1B and C), verifying the reliable culture system for erythropoiesis. The IFNg receptors, IFNGR1 (Fig. 1D) and IFNGR2 (Fig. 1E), had slight changes, maintaining relatively stable activity during differentiation, suggesting the potential openness of IFNg in erythropoiesis. Subsequently, the erythroid differentiation process in IFNg administration was continuously monitored. Primarily, cell proliferation increased in the first 2 days of culture (Fig. 1F, Supplementary Fig. 1), in line with the results of a study [16]. However, cell mitosis was suppressed in long-term treatment on day 8 (Fig. 1G), in line with another result [8]. Despite the paradoxical influence on cell proliferation, the differentiation process was remarkably accelerated by IFNg (Fig. 1H). One representative experiment was conducted, as shown in Fig. 1I, exhibiting the substantial cell population shift from CD235- to CD235þ. Besides the differentiation process was accelerated at the cellular level, the action was also hastened at the gene expression level (Supplementary Fig. 2). The erythroid-specific genes HBA1/2 were undoubtedly activated (Fig. 1J), while the transcriptional factor GATA1 was mildly promoted (Fig. 1K), implying some additional mechanism partly independent of the classic erythroid network was involved in the differentiation acceleration. Finally, the blocking effect of IFNg on commitment was also evaluated, revealing that the three commitments were all inhibited, especially BFU-E cells (Fig. 1L). 3.2. IFNg forcefully initiated early differentiation via an EPOindependent pathway IFNg potently pushed cells into the CD71þCD235þ population, accelerating the differentiation process toward erythrocytes. However, it was unclear whether this drive will still occur or stall if the crucial power EPO was withdrawn. To verify this, a similar culture system was used, with or without IFNg application (Fig. 2A). Unexpectedly, IFNg can still forcefully initiate early differentiation even without the existence of EPO. The CD71þCD235- and CD71þCD235þ population in IFNg administration was strikingly enhanced on day 4 (Fig. 2B and C). Besides 100 ng/mL IFNg, a lower concentration was also applied. The representative FACS results were shown in Fig. 2D, from which the 20 ng/mL group obtained a similar but slightly softer result. Furthermore, critical receptors for erythropoiesis were examined. Interestingly, c-KIT was downregulated (Fig. 2E), while EPOR was upregulated (Fig. 2F), indicating the opened differentiation access. Moreover, HBA1/2 and GATA1 were both upregulated (Supplementary Fig. 3A and B). All the
Please cite this article as: W. Wang et al., Interferon-g exerts dual functions on human erythropoiesis via interferon regulatory factor 1 signal pathway, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.068
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Fig. 1. IFNg significantly accelerated the in vitro erythroid differentiation process. (A) Flow chart displayed the in vitro culture program for erythropoiesis. (B-C) HBA1/2 globin (B) and EPOR (C) genes were monitored to confirm successful differentiation. Cells at checkpoints were collected and disposed of for RT Q-PCR. Statistical differences were analyzed from biological repeats. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (D-E) Relative expression abundance of IFNg receptors, IFNGR1 (D) and IFNGR2 (E), was evaluated. (F-G) Cell proliferation was calculated in short-term (F) and long-term (G) culture with 100 ng/mL IFNg treatment. (H) The CD71þCD235þ population on day 6 was statistically analyzed. FACS was performed by labeling cells with indicated antibodies. (I) The FACS results of one representative experiment was shown. 100 ng/mL IFNg was added in the liquid phase, and cells were collected and subsequently labeled with antibodies as indicated at each checkpoint. (J-K) RT Q-PCR was performed to monitor erythroid gene expression (J, HBA1/2; K, GATA1) under IFNg exertion on day 6. (L) CFC assay was performed to confirm the reported effect of IFNg on lineage commitment. After the 6-day pre-expansion, a total of 1000 cells were seeded into H4434 methylcellulose semi-solid medium (containing SCF, IL-3, EPO, and GM-CSF). Meanwhile, 100 ng/ml IFNg or PBS were added into the medium for the 14-day CFC formation. Then the mature colonies were characterized via microscopic examination.
abovementioned results suggested that IFNg was responsible for the stressful initiation of early erythropoiesis via an EPOindependent mechanism.
3.3. The transcription profile of hematopoietic cells was remodeled by IFNg administration Results had revealed a novel function of IFNg on erythropoiesis,
even strongly priming the early differentiation independent of EPO. However, the action of IFNg and involved mechanisms were still unclear. To determine these, a high-throughput RNAseq assay was performed. Four groups were involved, and the total transcription profile was presented as a heatmap (Fig. 3A). The two IFNg-incubated groups showed a resembling expression pattern, significantly distinct from the untreated ones. Considering the equal influence of IFNg on days 2 and 4 (Fig. 2D), it was reasonable that the common-
Please cite this article as: W. Wang et al., Interferon-g exerts dual functions on human erythropoiesis via interferon regulatory factor 1 signal pathway, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.068
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Fig. 2. IFNg initiated early erythroid differentiation independent of EPO. (A) The flow chart displayed the pre-differentiation culture system without EPO. (B-C) FACS results on day 4 were statistically analyzed, including the CD71þCD235- population (B) and CD71þCD235þ population (C). (D) One representative experiment was presented, including the day 2 and 4 results in 20 or 100 ng/mL IFNg administration. (E-F) RT Q-PCR determined the expression change of c-KIT (E) and EPOR (F) on day 4 with 100 ng/mL IFNg application.
regulated 453 genes were assumed as the potential pool for mechanism excavation (Fig. 3B). Next, GEO classifications and GSEA analyses were performed. The interferon signal pathway was profoundly enriched in the IFNg day 4 sample (Fig. 3C), similar to the GSEA results (Supplementary Fig. 3C). The heme metabolism was especially enriched (Supplementary Fig. 3D), implying the potential role of IFNg on hemoglobin production. Notably, the top upregulated genes were presented, among which IRF1 was noted (Fig. 3D, Supplementary Table 2). Moreover, the IRF family, IRF1 to IRF9, were further determined by RT Q-PCR, revealing that IRF1 was indeed the most motivated gene and had the highest abundance (Fig. 3E), suggesting IRF1 as the most reasonable effector for IFNg signal.
the gene transcription was also modulated. Both HBA1/2 (Fig. 4F) and GATA1 (Fig. 4G) were upregulated in IRF1 administration. Considering the slight change in SPI1 expression with IFNg administration (RNAseq data), it was reasonable that the IFNgeIRF1 axis might function through a pathway partly without SPI1. Due to the abnormal activation of IFNg in certain blood disorders, such as AA [6,11,17], its effector IRF1 also needs to be determined in related patients. The results revealed that IRF1 was activated in patients with AA (Fig. 4H), compared to healthy donors and those with myelodysplastic syndrome. All the abovementioned data proposed a novel hypothesis that the IFNgeIRF1 axis negatively blocked BFUE at the commitment stage and significantly accelerated the differentiation process at the later stage, leading to cleared erythropoietic windows and ultimately causing anemia (Fig. 4I).
3.4. IRF1 overexpression played dual functions on erythropoiesis just like IFNg
4. Discussion
To assess the latent value of IRF1, the overexpression of IRF1 was achieved using an optimized lentiviral vector (Supplementary Fig. 4). The infected primary cells were put into puromycin selection and EPO-driven differentiation, or CFC-assay as shown in Fig. 4A. The IRF1 enhanced group formed smaller and fewer colonies while the BFU-E colonies were more affected (Fig. 4B), indicating the blocking role of IRF1 on commitment. Subsequently, IRF1 performance on the erythroid process was further analyzed. First, IRF1 enforcement was confirmed by RT Q-PCR (Fig. 4C). Then, the erythropoietic process was continuously monitored. Surprisingly, ectopic IRF1 overexpression led to differentiation acceleration (Fig. 4D). The statistical analysis showed that IRF1 could powerfully and reproducibly augment the CD71þCD235þ population (Fig. 4E). Moreover, not only was the cell characteristic remodeled by IRF1,
A few studies have explored the impact of IFNg on erythropoiesis. It was of interest that the early-stage CD71þ population increased in mice under chronic IFNg accumulation, but with a lower output of erythrocytes [8,18]. In fact, it was reasonable to recognize this phenomenon as a compensatory consequence of the increased EPO, raising corrective feedback to anemia. In this study, a more accurate strategy was performed in vitro, and a systematic conclusion was deduced. To our knowledge, it was the first time that IFNg was found to accelerate the differentiation process as a dual regulator, leading to erythropoietic window clearance, and the dual influence of IRF1 described here was also unreported previously. Interestingly, except IFNg, TGFb was also found to play comparatively similar functions, not only reducing BFU-E proliferation but also accelerating downstream differentiation [19],
Please cite this article as: W. Wang et al., Interferon-g exerts dual functions on human erythropoiesis via interferon regulatory factor 1 signal pathway, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.068
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Fig. 3. Bioinformatical analysis excavated the molecular mechanism of IFNg in differentiation acceleration. (A) The RNAseq transcription profile of the 4 groups was shown in the heatmap. The log10 (FPKMþ1) values were normalized and clustered. The red color indicated high expression, while the blue color indicated low expression. (B) The Wayne diagram showed the overlap of different genes among different comparison combinations. The numbers in the circle represent the sum of both upregulated (2-fold) and downregulated (0.5-fold) genes in the comparative combinations. (C) As a result of GO enrichment analysis, the most significant 20 terms were selected to draw a histogram. It can determine which biological functions or pathways were significantly enriched. (D) The top 8 upregulated trans factors in IFNg administration were arranged in table. (E) The IRF family was systematically scanned by RT Q-PCR to select the exact member for IFNg signal. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
indicating the resembling functions of these negative cell factors on hematopoiesis. The cytokine receptors, signal adaptors, and trans factors were all considered in the mechanism. Instead of c-KIT and EPOR being suppressed as previously reported [20], EPOR was obviously activated in our study, consistent with the differentiation acceleration. Moreover, a similar difference in GATA1 between another study [21] and our work was also noted. This contradiction may be partially explained by time, because cells may adjust their response to IFNg stimulation with time. For instance, cell proliferation was efficiently enhanced in short time culture, coincident with some results [16,22], but strongly impaired in long term culture, similar to other findings [8]. Thus, the temporary stress and long term influence would switch diversely, suggesting the time-axis significance of IFNg in erythropoiesis. It was confirmed that several vital signals were involved in IFNg-administrated erythropoiesis. STAT1, the critical effector of IFNg signal, and JAK2, the essential adaptor of EPO signal, were both dramatically stimulated (Supplementary Table 2), similar to the protein modification previously described [17]. Stat1 favored megakaryopoiesis and inhibited erythropoiesis [23,24], which slightly contradicts the result of this study, but it was also reported that EPO could similarly activate Stat1 as downstream signal [25],
implying the complicated role of Stat1 in erythropoiesis. Interestingly, Stat1-deficient erythroid cells revealed a less differentiated phenotype [26], implying its potential regulatory role on erythropoiesis, consistent with the upregulated STAT1 expression along the accelerated differentiation. However, the upregulation of STAT1 may be partially due to its self-control feedback [27]. It was also unlikely that IFNg mainly functioned through the JAK2 pathway because all downstream erythroid-specific trans factors were only softly modulated or had slight changes. In this study, we instead paid more attention to resurvey the remodeling of transcriptional network. Based on high-throughput RNAseq data, IRF1, a crucial immunological protein against viral and bacterial infection [8,28], was identified. Unlike earlier findings that IRF1 controlled granulopoiesis instead of erythropoiesis [29] or coordinated with SPI1 as the inhibitor of erythropoiesis [8], it was revealed in our survey as an energetic speeder for erythroid differentiation, similar to IFNg. Recently, the IFNgeIRF1 axis is still concerned in hematopoiesis and blood disorders [6,11,16,30e33]. Some abnormal IFNg expression was even detected in the plasma of patients with polycythemia vera, implying the likely feedback of IFNg on increased erythropoiesis [34]. Our findings may outline a beneficial principle for the IFNgeIRF1 axis on hematopoiesis and endow IFNg new potential value for blood disorder therapy.
Please cite this article as: W. Wang et al., Interferon-g exerts dual functions on human erythropoiesis via interferon regulatory factor 1 signal pathway, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.068
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Fig. 4. Ectopic IRF1 overexpression played dual functions in erythropoiesis. (A) The vector construction (upper) and culture flow (lower) were presented. The lentiviral vector carrying sfGFP alone without IRF1 was used as a control vector, and both groups were selected under 0.5 mg/mL puromycin. (B) The colony assay under IRF1 overexpression was performed. After transducted for 3 days, approximately 500 transduced primary cells were seeded in H4434 medium and meanwhile supplied with 0.1 mg/mL puromycin. After 14day culture, the formatted colonies were counted. (C) RT Q-PCR was performed to determine ectopic IRF1 expression in primary cells. Cells on day 2 were collected and disposed of for RT Q-PCR. (D) The cellular differentiation process was monitored, and representative results were arranged. Cells were collected, stained and detected by FACS. (E) Biological repeats were also executed to gain statistical results. (F-G) The expression pattern of HBA1/2 (F) and GATA1 (G) were examined by RT Q-PCR. Cultured cells on day 5 were gathered for RT Q-PCR. (H) IRF1 expression was assessed in peripheral blood mononuclear cells (PBMCs) from normal healthy donors and patients. The PBMCs were separated by Ficoll density centrifugation, with subsequent RT Q-PCR. (I) A novel IFNgeIRF1 function model was hypothesized. IFNgeIRF1 axis may play two disparate effects on erythropoiesis, blocking erythroid lineage commitment and accelerating erythroid differentiation process, leading to erythropoietic window clearance.
Therefore, this study systematically explored the potential function of IFNg in erythropoiesis and proposed a novel hypothesis to demonstrate the dual functions of the IFNgeIRF1 axis in erythropoiesis, which may place valuable implications for understanding and treatment of erythroid-related diseases. Disclosure of conflicts of interest
(19JCZDJC33000, 18JCQNJC11900); The Beijing-Tianjin-Hebei basic research project (18JCZDJC44600/H2018206423); Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (2017PT31047, 2018PT31038, 2018PT32028); CAMS Innovation Fund for Medical Sciences (2016-I2M-1-018, 2017-I2M1-015); Novo Nordisk Haemophilia Research Fund in China, Denmark. We thank Yahong You for supplying several blood samples from patients.
All the authors declare no competing financial interests. Acknowledgements Appendix A. Supplementary data This work was supported by National Natural Science Foundation of China (81500084, 81970121, 81600099, 81900126); Tianjin Municipal Science and Technology Commission Grant
Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.10.068.
Please cite this article as: W. Wang et al., Interferon-g exerts dual functions on human erythropoiesis via interferon regulatory factor 1 signal pathway, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.068
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Please cite this article as: W. Wang et al., Interferon-g exerts dual functions on human erythropoiesis via interferon regulatory factor 1 signal pathway, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.068