Abstract
Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome characterized by overwhelming immune activation. A steroid and chemotherapy-based regimen remains as the first-line of therapy but it has substantial morbidity. Thus, novel, less toxic therapy for HLH is urgently needed. Although differences exist between familial HLH (FHL) and secondary HLH (sHLH), they have many common features. Using bioinformatic analysis with FHL and systemic juvenile idiopathic arthritis, which is associated with sHLH, we identified a common hypoxia-inducible factor 1A (HIF1A) signature. Furthermore, HIF1A protein levels were found to be elevated in the lymphocytic choriomeningitis virus infected Prf1−/− mouse FHL model and the CpG oligodeoxynucleotide-treated mouse sHLH model. To determine the role of HIF1A in HLH, a transgenic mouse with an inducible expression of HIF1A/ARNT proteins in hematopoietic cells was generated, which caused lethal HLH-like phenotypes: severe anemia, thrombocytopenia, splenomegaly, and multi-organ failure upon HIF1A induction. Mechanistically, these mice show type 1 polarized macrophages and dysregulated natural killler cells. The HLH-like phenotypes in this mouse model are independent of both adaptive immunity and interferon-γ, suggesting that HIF1A is downstream of immune activation in HLH. In conclusion, our data reveal that HIF1A signaling is a critical mediator for HLH and could be a novel therapeutic target for this syndrome.Introduction
Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of overwhelming immune activation characterized by several clinical features, such as high fever, multi-lineage cytopenia, splenomegaly, and hyperferritinemia.31 In primary HLH patients, various mutations in genes related to the granule-dependent cytotoxicity pathway in T/natural killer (NK) cells have been identified.4 CD8 T cells and interferon gamma (IFN-γ) have been shown to play critical roles in the pathogenesis of primary HLH, the onset of which is usually triggered by viral infection.5 On the other hand, secondary HLH (sHLH) has a heterogeneous etiology, which is often triggered by systemic viral infection, autoimmune disorder, or hematologic malignancies.63 In contrast to primary HLH, cytotoxic activity of T/NK cells is not always decreased in sHLH.7 Macrophage activation syndrome (MAS), a form of sHLH in the context of rheumatic disease that is especially associated with systemic juvenile idiopathic arthritis (sJIA),8 is associated with aberrant toll-like receptor (TLR)-induced gene expression patterns.9 Despite reports that primary and sHLH have different genetic aberrations and distinct cytotoxic activities, the key features for HLH (cytopenias and a unique presentation of extreme inflammation) remain common.7 Thus, we hypothesized that there are common underlying downstream mediators for HLH phenotype development. Identifying these mediators for HLH will not only help to understand the disease, but also lead to the development of better therapies.
Hypoxia-inducible factor (HIF), which was originally discovered as a critical transcription factor for optimal cellular adaptation to hypoxia, actually plays an important role in immune response, both in hypoxia and normoxia conditions1110 HIF consists of heterodimers, HIF-α subunit and ARNT. ARNT is stably expressed in many cell types, while HIF-α subunits are expressed differently in tissues and cells. So far, three isoforms of HIF-α subunits, HIF1A, HIF2A, and HIF3A, have been documented.12 HIF1A is widely expressed in both innate and adaptive immune cells,1613 while HIF2A/EPAS1 expression is limited in certain immune cell types,1817 and the HIF3A expression pattern has not been so well studied. Many stimuli or factors that have a function in the immune response lead to HIF1A protein accumulation and activation independent of the hypoxic regulation. Bacteria, lipopolysaccharide (LPS), and tumor necrosis factor-α (TNF-α) have been well documented to induce HIF1A accumulation in macrophages, thereby boosting their microbicidal capacity.2019 T-cell receptor ligation in T cells increases HIF1A transcription and HIF1A protein accumulation. Moreover, cytokines, such as IL-2, induce HIF1A accumulation in CD8 T cells enhancing their cytotoxic function.10 There is growing evidence to indicate the potential role of HIF1A signaling in immune activation.
To identify the key downstream mediators for HLH, we first performed a bioinformatic analysis of published microarray expression datasets of familial hemophagocytic lymphohistiocytosis (FHL) and sJIA.219 We found that the HIF1A signature, which refers to a group of HIF1A-induced genes, was enriched in both FHL and sJIA patients’ peripheral blood mononuclear cells (PBMCs). Then, we confirmed that HIF1A protein expression was significantly increased in two widely-used HLH mouse models. Furthermore, in vivo data from transgenic mice show that activation of HIF1A in hematopoietic cells results in HLH-like phenotypes. Our study suggests that the HIF1A signaling pathway is a critical pathological downstream mediator for HLH development.
Methods
Cell line and mice
Murine cell line Raw264.7 was purchased from ATCC. Cells were cultured in DMEM medium in a 5% CO2 incubator at 37°C, and subcultured every 2–3 days. Rag1/, Ifngr/, Ifng/, Prf1/, Vav1-Cre, and Rosa26-LSL-rtTA mice were purchased from Jackson Laboratory. Transgenic HIF1A-TPM mice were kindly provided by Professor James Bridge from Cincinnati Children’s Hospital Medical Center (CCHMC).22 Mice, with the genotype of Ncr1-iCre, were kindly provided by Professor Eric Vivier from the French INSERM Laboratory.23 LCMV-infected Prf1/ mouse model and the repeated CpG-treated mouse model were generated as previously described.25245 All animal studies were performed according to an approved Institutional Animal Care and Use Committee protocol and federal regulations.
Statistical analysis
Data were analyzed by Prism 6.0 (GraphPad Software). P<0.05 was considered significant. Continuous variables were analyzed by using Student t-test or one-way ANOVA. Mice survival was estimated using the Kaplan-Meier method.
Information concerning the antibodies and reagents, bioinformatic analysis, flow cytometry, ELISA, western blot, histology, generation of bone marrow-derived macrophages, and the assay used to determine the capacity of macrophages to engulf erythroblasts is reported in detail in the Online Supplementary Appendix.
Results
Activated HIF1A signaling in FHL and sJIA patients
Immune activation is coupled with cytokine signaling and transcriptional changes. To investigate the network of transcription factors in HLH pathogenesis, we took a bioinformatic approach and analyzed two published microarray datasets of patients with FHL and sJIA.219 Since there are no available sHLH transcript profile data, we utilized the microarray data of sJIA patients which are more likely to be complicated by macrophage activation syndrome, a subtype of sHLH in the context of rheumatoid diseases.2826 We performed unbiased TF-target enrichment analysis29 and found that HIF1A, NF-κB, GATA1, and STAT1 are the common immune-related transcription factors in both the FHL and sJIA datasets (Figure 1A and B and Online Supplementary Figure S1A and B). Among these transcription factors, HIF1A is of particular interest as it regulates not only the up-regulated genes but also the down-regulated genes in HLH, indicating that HIF1A might be a critical mediator in HLH development. HIF1A is known to be regulated in both transcriptional and post-translational levels. In the FHL dataset, HIF1A mRNA is increased in the FHL patients compared to healthy donors; however, there is no significant difference in HIF1A expression between FHL patients with and those without a genetic diagnosis (Online Supplementary Figure S1C and D). At the same time, in the sJIA dataset, there is no significant change in HIF1A expression at the mRNA level between patients and healthy donors (Online Supplementary Figure S1E).
To investigate enrichment of the HIF1A signature in HLH, we also utilized another bioinformatic approach of gene set enrichment analysis (GSEA) to analyze these two datasets, and revealed that the HIF1A signature is significantly enriched in both the FHL and sJIA PBMCs datasets (Figure 1C and D). However, there is no significant difference in HIF1A signature enrichment between FHL patients with and those without a genetic diagnosis (Online Supplementary Figure S1F). There are 258 leading edge genes (LEGs) in the FHL dataset and 214 LEGs in the sJIA dataset with 108 overlapping common LEGs (Figure 1E). Gene ontology analysis showed that these overlapping common LEGs are related to blood coagulation, chemotaxis, glycolysis, oxygen species metabolic process, platelet activation, immune response, and cytokines (Figure 1E). These results further suggest that HIF1A may play a key role in regulating downstream targets in both primary HLH and sHLH patients.
Elevated HIF1A protein in LCMV-infected Prf1−/− and CpG-treated mouse HLH models
Several mouse models that recapitulate primary HLH, sHLH, or MAS have been reported.7 We determined whether HIF1A signaling is activated in established HLH mouse models. The LCMV-infected perforin-deficient (Prf1/) mouse model is a well-known HLH mouse model that recapitulates biallelic perforin mutation patients with pathogen infection (Figure 2A).5 After challenging them with LCMV, the Prf1/ mice quickly developed anemia and thrombocytopenia (Figure 2B). CD8 T cells activate macrophages via secreting IFN-γ in this mouse model. We measured HIF1A expression levels in Gr1CD115F4/80SSC spleen macrophages by using flow cytometry (Figure 2C)30 and found that HIF1A levels in spleen macrophages were significantly increased in LCMV-infected Prf1/ mice compared to the control mice (Figure 2D). We also identified profound type 1 polarized macrophages in the spleen (Figure 2E and F) and bone marrow (data not shown) from the LCMV-infected Prf1/ mice.
Repeated injections of TLR9 ligand CpG oligodeoxynucleotides (ODN) causes sHLH in wild-type (WT) mice,24 which mimics sHLH features (Figure 2G). After injecting CpG five times into WT mice, CpG-treated-mice developed anemia and thrombocytopenia (Figure 2H). Similar to what was observed in the primary HLH model, we found that HIF1A expression in the spleen macrophages was also increased in CpG-treated mice (Figure 2I). Type 1 polarization of macrophages in spleen (Figure 2J and K) and bone marrow (data not shown) was observed in CpG-treated mice. Taken together, these data suggest that HIF1A protein expression is elevated both in primary HLH and sHLH mouse models.
Inducible activation of HIF1A is sufficient for developing HLH-like phenotypes in C57BL/6 background mice
To determine the significance of HIF1A signaling activation in HLH development in vivo, we generated transgenic mice with inducible HIF1A/ARNT expression in hematopoietic cells. We combined the Vav1-Cre allele, Rosa26-loxp-stop-loxp (LSL) reverse-tetracycline-controlled transactivator (rtTA) allele, and triple point mutation (TPM) HIF1A/wild-type ARNT alleles (tet-on-TPM/ARNT) (Vav1-Cre/TPM). Triple point mutations include P402A, P564A and N803A, which prevent degradation and facilitate transcriptional activation of HIF1A.22 Thus, Vav1-Cre/TPM mice have stabilized and constitutively active HIF1A protein (Figure 3A) in hematopoietic cells after administration of doxycycline. Vav1-Cre mice without the TPM allele (Vav1-Cre/WT) served as control. We confirmed an increase in HIF1A protein level in both c-Kit positive and negative cells (Figure 3B) by western blot and in individual cell lineages by flow cytometry (Figure 3C) in Vav1-Cre/TPM mice compared to control mice. Importantly, the level of HIF1A in macrophages in TPM mice is comparable to that in CpG-injected HLH mice (Online Supplementary Figure S2).
After doxycycline administration, TPM mice with the pure C57BL/6 background quickly developed severe anemia and thrombocytopenia (Figure 3D and E). Bone marrow cellularity was dramatically reduced in the TPM mice (Figure 3F and Online Supplementary Figure S3A). We did not find a blockade of erythropoiesis in the bone marrow and spleen from Vav1-Cre/TPM mice (Online Supplementary Figure S4), indicating a cell extrinsic mechanism for quick progression of anemia and a decrease in bone marrow cellularity. Consistent with the diagnostic criteria for HLH, TPM mice showed splenomegaly (Figure 3G and H). Normal splenic follicular architecture was disrupted in the TPM mice (Online Supplementary Figure S3B). Liver dysfunction is commonly observed in HLH patients. Indeed, TPM mice had substantial inflammatory cells infiltrated into the liver (Figure 3I). Flow cytometric analysis revealed that most of these cells were CD11b myeloid cells (Figure 3J). However, we failed to find robust hemophagocytosis in the cytospins or sections of bone marrow, spleen, or liver (data not shown). High levels of serum ferritin, which is also one of the diagnostic criteria for HLH, was observed in the TPM mice in comparison with the control mice (Figure 3K). Furthermore, several inflammatory cytokines, such as IL-6, IL-12, and IFN-γ, were increased in the serum from the TPM mice (Online Supplementary Figure S5). All of the mice succumbed within three weeks (Figure 3L). We further confirmed that several genes related to chemokine, macrophage activation, and glycolysis (which are the common LEGs of HIF1A signature in FHL and sJIA datasets), were elevated in the Vav1-Cre/TPM mice (Figure 3M and Online Supplementary Figure S6). Taken together, inducible expression of stabilized and active HIF1A with ARNT gives rise to HLH-like phenotypes in pure C57BL/6 background mice.
Unaffected T-cell populations and dysregulated NK cells in induced TPM mice
Given that robust activation of CD8 T cells is observed in the primary HLH mouse model, we first determined the T-cell populations in TPM mice. However, no significant change in the frequency and absolute number of CD8 T cells was observed in the TPM mice (Figure 4A and B). We also measured IFN-γ production in CD8 T cells and did not find a significant difference between the TPM mice and the control mice (Figure 4C), indicating that CD8 T cells may not play a key role in HIF1A induced HLH-like phenotypes.
Natural killer cell activity is important for immune homeostasis. NK cell defect is one of the critical features of primary HLH. Reduced NK cell number or impaired NK cell function has been reported in some of the sHLH patients. Interestingly, we found a significant reduction in the number of total NK cells and DX5 mature NK cells in the spleen (Figure 4D–F), peripheral blood and bone marrow (data not shown) from the TPM mice. Importantly, IFN-γ production in NK cells was impaired in the TPM mice (Figure 4G). However, cell surface CD107a expression in NK cells was comparable between the TPM mice and the control mice (Figure 4H), suggesting no major defect in degranulation of cytotoxic granules in TPM mice. It has been reported that hypoxia may lead to a reduction in NKp46 expression, an NK cell activating receptor, in vitro.31 However, there was no significant difference in NKp46 expression between the TPM mice and the control mice (Figure 4I). These data suggest that TPM mice have quantitative and functional dysregulation in NK cells.
To determine whether the impairment of NK cells is due to intrinsic or extrinsic NK cell factors, we generated Ncr1-iCre/LSL/TPM (Ncr1-iCre/TPM) mice. Using GFP reporter, we confirmed that Ncr1-iCre is specifically expressed in NK cells (Online Supplementary Figure S7A and C).23 Surprisingly, after NK cell specific TPM induction, we did not find any changes in the NK cell number or differentiation pattern compared to the control mice (Online Supplementary Figure S7B and D), indicating that the NK cell dysregulation in Vav1-Cre/TPM mice may be due to a non-autonomous cellular mechanism.
Slightly changed dendritic cells but strongly polarized Type 1 macrophages in induced TPM mice
Since a minor fraction of dendritic cells (DCs) persistently present antigens and drive T cells in the primary HLH mouse model, we measured DC population in the TPM mice. There was an increase in the number of plasmacytoid DCs (pDCs) in the early stage but not in the number of conventional DCs (cDCs) (Figure 5A–C). However, at a later time, there was no significant difference in numbers of pDCs and cDCs between the TPM mice and the control mice (Online Supplementary Figure S8).
Macrophage activation by diverse triggers is a common feature in HLH. We found type 1 polarization of macrophages in both primary and sHLH models; thus, we investigated the macrophage population in TPM mice. The number of macrophages was significantly increased in the spleen, but not in the bone marrow in TPM mice (Figure 5D and G). More importantly, the macrophages were polarized toward type 1 in both bone marrow and spleen from the TPM mice (Figure 5E, F, H and I). To further determine whether type 1 polarized macrophages are able to phagocytose erythrocytes and cause anemia, we cultured erythroblasts with IFN-γ-polarized type 1 bone marrow-derived macrophages (BMDMs) or IL-4-polarized type 2 BMDMs and found that only type 1 macrophages, but not type 2 macrophages, engulfed erythroblasts (Figure 5J–L and Online Supplementary Figure S9). Taken together, our data suggest that HIF1A signaling activation causes type 1 macrophage polarization, which might also contribute to engulfment of erythroblasts and cause anemia in the HLH disease scenario.
Adaptive immune cells are not required for TPM-induced HLH phenotypes
Since there was no change in the frequency of the total and IFN-γ producing CD8 T cells in the TPM mice, we further investigated the role of the lymphocytes in TPM-induced HLH phenotypes. We crossed Vav1-Cre/TPM mice with recombination activation gene 1 (Rag1)-deficient (Rag1−/−/) mice that lack T cells and B cells (Figure 6A B) and generated Rag1−/−/Vav1-Cre/TPM mice. Rag1−/−/Vav1-Cre/WT mice served as control. After administration of doxycycline, Rag1−/−/Vav1-Cre/TPM developed similar anemia, thrombocytopenia, and splenomegaly as the Vav1-Cre/TPM mice (Figure 6C and D). Type 1 macrophage polarization was also observed in Rag1−/−/Vav1-Cre/TPM mice (Figure 6E). Survival of Rag1−/−/Vav1-Cre/TPM mice was not prolonged compared to Vav1-Cre/TPM mice (Figure 6F). These data indicate that adaptive immunity is not essential for TPM-induced HLH phenotypes and non-lymphoid cells are sufficient to mediate disease progression in TPM mice.
Genetically blocking IFN-γ signaling could not rescue TPM-induced phenotypes except to partly rescue the anemia
IFN-γ is a critical factor upstream of HIF1A for type 1 macrophage polarization and is essential for disease development in the LCMV-infected Prf1−/− HLH mouse model. Thus, we determined the role of IFN-γ signaling in TPM-induced HLH. We generated Ifngr−/−/Vav1-Cre/TPM (Ifngr−/−/TPM) mice. Ifng−/−/Vav1-Cre/WT (Ifngr−/−/WT) mice served as control. Interestingly, TPM-induced anemia was partially rescued in Ifngr deficient mice (Figure 7A). However, the Ifngr−/−/TPM mice still developed severe thrombocytopenia (Figure 7B) and all of them succumbed to disease, but with a prolonged latency compared to the Vav1-Cre/TPM mice (Figure 7C). Flow cytometric analysis revealed that TPM-induced type 1 polarization of macrophages was not blocked in Ifngr-deficient mice (Figure 7D). We also generated Ifng−/−/Vav1-Cre/TPM (Ifngr−/−/TPM) mice and found similar results (Online Supplementary Figure S10) indicating that HIF1A-induced HLH-like phenotypes in TPM mice are independent of IFN-γ ligand and receptor. It is likely that IFN-γ ligand and receptor are the upstream of HIF1A signaling, and HIF1A activation itself could lead to type 1 macrophage polarization to some degree even without IFN-γ ligand and receptor. To determine whether IFN-γ could induce HIF1A signaling and cause type 1 polarization in macrophages, we treated the mouse macrophage cell line, Raw264.7 cells, with IFN-γ. We found that the level of HIF1A protein and the expression of known HIF1A target genes, including the critical macrophage polarization gene Nos2, are significantly increased in the IFN-γ treated Raw264.7 cells compared to the control (Figure 7E and F). We also cultured BMDMs from TPM mice and induced TPM expression in vitro. We found an increase in mRNA expression of macrophage polarization-related gene (Nos2), glycolysis-related genes (Hk2, Pfkfb3), and also other HIF1A direct target genes (Adm) (Figure 7G and H). These genes were similarly activated by IFN-γ when treated with Raw264.7 cells (Figure 7F). The evidence suggests that HIF1A signaling is the downstream of IFN-γ signaling and that activation of HIF1A signaling could activate and polarize macrophages which results in some key features of HLH-like phenotypes. Activation of HIF1A signaling in combination with activation of other pathways, such as NF-κB and STAT1, might lead to the complete complex HLH phenotypes as seen in humans (Figure 7I).
Discussion
Although there are differences in etiology and pathological immune response between FHL patients and sHLH patients, they have similar clinical manifestations and the same features of hyper-inflammation and hyper-immune response.72 Identification of key common mediators in HLH may help to explore novel therapeutic targets that would have a wider application in HLH patients. In the present study, we identified that the HIF1A signature is enriched in both FHL and sJIA patients, and its protein level is elevated in primary and sHLH mouse models. Induction of HIF1A signaling in hematopoietic cells in vivo results in HLH-like phenotypes. This indicates that HIF1A is a critical mediator for HLH.
HIF1A is reported to be involved in inflammation and immune response.321210 In line with this, our bioinformatic analysis of FHL and sJIA datasets revealed that HIF1A might have a wide regulatory effect in HLH pathogenesis, which may be related to regulation of chemotaxis, cytokines, immune response, glycolysis, blood coagulation, and apoptosis, as indicated by the GO analysis. Notably, it is evident that FHL patients have a stronger signature than sJIA patients. This could be due to the independent processes of these two array datasets from both groups. There is also a possibility that the strong genetic component of FHL leads to this discrepancy. However, we did not observe a significant difference in the HIF1A signature between FHL patients with and those without a genetic diagnosis in this dataset. It is hard to completely rule out the possibility that genetic mutation drives HIF1A signature since only mutations of PRF1, UNC13D, and STX11 were tested in this FHL microarray dataset.21 Patients without a genetic diagnosis may still carry disease-causing mutations. Future studies are needed to clarify whether the strong genetic background could have additional effects on the gene signatures. Nonetheless, the stronger signature in FHL patients as compared to the sJIA patients could be the underlying difference between these two diseases, and it is possible that the involvement of distinct cell types causes this difference. T cells, NK cells, macrophages, and DCs are all involved in the FHL pathogenesis, while T cells and NK cells are less involved in the sJIA pathogenesis.33 Thus, in microarray datasets of PBMCs, FHL may show a stronger signature than sJIA.
Macrophage activation/polarization is a common feature in HLH mouse models irrespective of their specific etiology. Macrophages have been reported to switch their metabolism from oxidative phosphorylation towards glycolysis upon pro-inflammatory stimuli by the upregulation of HIF1A.3534 Indeed, we found that HIF1A was stabilized in macrophages in both the LCMV/Prf1−/− model and the CpG model. Our data are consistent with other reports that numerous stimuli, such as IFN-γ, TNF-α, CpG, and LPS, are able to increase the HIF1A protein level in macrophages.10 These cytokines and TLR ligation might co-operate to increase HIF1A protein levels in HLH.
Although several studies have showed an HIF1A deficiency in myeloid cells leads to impaired inflammatory responses, the effect of activation of HIF1A in hematopoietic cells in vivo remains unclear. Here, we show that induction of HIF1A in hematopoietic cells in vivo is lethal and gives rise to some HLH-like phenotypes, such as severe anemia, thrombocytopenia, splenomegaly, liver damage, ferritinemia, and macrophage activation, suggesting that HIF1A is a critical mediator in HLH. We are also aware that HIF1A-induced phenotypes cannot recapitulate all the manifestations seen in human HLH patients. This could be due to the fact that other transcription factors which co-operate with HIF1A activation to generate the overt HLH phenotypes are required. Our analysis of the transcription factor network sheds light on other key transcription factors in HLH development which could help in future research.
Defective CD8 T cells are regarded as the driver in the primary HLH mouse model.5 However, the HLH-like phenotypes in the TPM mouse model are not dependent on lymphocytes since activation of HIF1A also causes HLH phenotypes in Rag1−/− mice. This indicates that non-lymphocytes contribute to the HLH-like phenotypes. Here, we observed that HIF1A activation leads to macrophage type 1 polarization in vivo. Our in vitro data and other reports revealed that HIF1A can up-regulate Nos2, IL-6, IL-1β, CXCR4 and, glycolysis-related genes which might account for the in vivo type 1 polarization.32 The role of type 1 polarization of macrophages for anemia in HLH is still unclear. Our in vitro data show that IFN-γ-polarized type 1 macrophages engulf erythroblasts, which is consistent with our earlier report36 that IFN-γ acts directly on macrophages resulting in hemophagocytosis, leading to a consumptive anemia in vivo. There is also evidence that hemophagocytes express type 2 polarized macrophage markers such as CD206 or CD163, and exhibit expression profiles similar to resting splenic macrophages.3837 This discrepancy in distinct macrophage activation type may be due to a different subpopulation of macrophage, or to various different etiological scenarios. Although IFN-γ-polarized type 1 macrophages phagocytose erythroblasts in our in vitro experiment setting, robust phagocytosis was not observed in the TPM mice, which suggests that type 1 polarization of macrophages induced by HIF1A transgene is not sufficient to induce hemophagocytosis in vivo.403924 Other additional factors, such as blocking IL-10 signaling or involvement of DCs may be required for phagocytosis in vivo. Future investigation will be needed to verify these possibilities.
IFN-γ is a potent stimulator for type 1 macrophage polarization, and plays a central role in a large proportion of HLH patients and in FHL mouse models; however, it has also been seen that it is not essential in some of the sHLH mouse models.4139 Our study also showed that TPM-induced HLH-like phenotypes are independent of IFN-γ. In fact, our in vitro data and other reports showed that HIF1A is a downstream effector of IFN-γ, and activation of HIF1A in macrophages leads to the increase in type 1 polarization-related genes, such as Nos2, and other glycolysis-related genes. However, partial rescue of anemia was observed in TPM mice with IFN-γ deficiency. IFN-γ may also have HIF1A-independent mechanisms that affect erythropoiesis, as has been reported.42
In summary, our study suggests that HIF1A is a common critical downstream mediator for HLH. We propose that HIF1A activation as the consequence of systemic inflammation, cytokine storm, or ligation of TLR may contribute to HLH development. Thus, HIF1A might be a promising therapeutic target for HLH intervention.
Acknowledgments
The authors would like to thank Eric VIVIER who kindly provided the transgenic mice.
Footnotes
- RH and YH contributed equally to this work and GH and YHL contributed equally to this study as joint senior authors
- Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/102/11/1956
- FundingThis work was supported by a Pilot Grant of The HLH Center of Excellence at Cincinnati Children’s Hospital Medical Center (to GH), and a grant from Histiocytosis Association Research Grant Program (to GH), Southern Medical University Basic Research Grant Program (No. QD2016N016 to RH), Natural Science Foundation of Guangdong Province, China (No. 2017A030310112 to RH), National Natural Science Funds of China (No. 81300392 to JW, No. 81370611 to ZFX, No. 81470338 to YZ, No. 81470297, and No. 81770129, to GH, and No. 81530008, and No. 81470295 to ZJX, No. 81570173 to XL), Tianjin science and technology projects (13JCYBJC42400 to YZ). We would like to acknowledge the assistance of the Research Flow Cytometry Core in the Division of Rheumatology at Cincinnati Children’s Hospital Medical Center. All flow cytometric data were acquired using equipment maintained by the Research Flow Cytometry Core.
- Received June 20, 2017.
- Accepted August 24, 2017.
References
- Janka GE, Lehmberg K. Hemophagocytic lymphohistiocytosis: pathogenesis and treatment. Hematology Am Soc Hematol Educ Program. 2013; 2013:605-611. PubMedhttps://doi.org/10.1182/asheducation-2013.1.605Google Scholar
- Jordan MB, Allen CE, Weitzman S, Filipovich AH, McClain KL. How I treat hemophagocytic lymphohistiocytosis. Blood. 2011; 118(15):4041-4052. PubMedhttps://doi.org/10.1182/blood-2011-03-278127Google Scholar
- Lehmberg K, Nichols KE, Henter JI. Consensus recommendations for the diagnosis and management of hemophagocytic lymphohistiocytosis associated with malignancies. Haematologica. 2015; 100(8):997-1004. PubMedhttps://doi.org/10.3324/haematol.2015.123562Google Scholar
- Filipovich AH, Chandrakasan S. Pathogenesis of Hemophagocytic Lymphohistiocytosis. Hematol Oncol Clin North Am. 2015; 29(5):895-902. Google Scholar
- Jordan MB, Hildeman D, Kappler J, Marrack P. An animal model of hemophagocytic lymphohistiocytosis (HLH): CD8+ T cells and interferon gamma are essential for the disorder. Blood. 2004; 104(3):735-743. PubMedhttps://doi.org/10.1182/blood-2003-10-3413Google Scholar
- Lehmberg K, Sprekels B, Nichols KE. Malignancy-associated haemophagocytic lymphohistiocytosis in children and adolescents. Br J Haematol. 2015; 170(4):539-549. Google Scholar
- Brisse E, Wouters CH, Matthys P. Advances in the pathogenesis of primary and secondary haemophagocytic lymphohistiocytosis: differences and similarities. Br J Haematol. 2016; 174(2):203-217. Google Scholar
- Emile JF, Abla O, Fraitag S. Revised classification of histiocytoses and neoplasms of the macrophage-dendritic cell lineages. Blood. 2016; 127(22):2672-2681. PubMedhttps://doi.org/10.1182/blood-2016-01-690636Google Scholar
- Fall N, Barnes M, Thornton S. Gene expression profiling of peripheral blood from patients with untreated new-onset systemic juvenile idiopathic arthritis reveals molecular heterogeneity that may predict macrophage activation syndrome. Arthritis Rheum. 2007; 56(11):3793-3804. PubMedhttps://doi.org/10.1002/art.22981Google Scholar
- Palazon A, Goldrath AW, Nizet V, Johnson RS. HIF transcription factors, inflammation, and immunity. Immunity. 2014; 41(4):518-528. PubMedhttps://doi.org/10.1016/j.immuni.2014.09.008Google Scholar
- Greer SN, Metcalf JL, Wang Y, Ohh M. The updated biology of hypoxia-inducible factor. EMBO J. 2012; 31(11):2448-2460. PubMedhttps://doi.org/10.1038/emboj.2012.125Google Scholar
- Cummins EP, Keogh CE, Crean D, Taylor CT. The role of HIF in immunity and inflammation. Mol Aspects Med. 2016;24-34. Google Scholar
- Cramer T, Yamanishi Y, Clausen BE. HIF-1alpha is essential for myeloid cell-mediated inflammation. Cell. 2003; 112(5):645-657. PubMedhttps://doi.org/10.1016/S0092-8674(03)00154-5Google Scholar
- Walmsley SR, Chilvers ER, Thompson AA. Prolyl hydroxylase 3 (PHD3) is essential for hypoxic regulation of neutrophilic inflammation in humans and mice. J Clin Invest. 2011; 121(3):1053-1063. PubMedhttps://doi.org/10.1172/JCI43273Google Scholar
- Jantsch J, Chakravortty D, Turza N. Hypoxia and hypoxia-inducible factor-1 alpha modulate lipopolysaccharide-induced dendritic cell activation and function. J Immunol. 2008; 180(7):4697-4705. PubMedhttps://doi.org/10.4049/jimmunol.180.7.4697Google Scholar
- McNamee EN, Korns Johnson D, Homann D, Clambey ET. Hypoxia and hypoxia-inducible factors as regulators of T cell development, differentiation, and function. Immunol Res. 2013; 55(1–3):58-70. PubMedhttps://doi.org/10.1007/s12026-012-8349-8Google Scholar
- Imtiyaz HZ, Williams EP, Hickey MM. Hypoxia-inducible factor 2alpha regulates macrophage function in mouse models of acute and tumor inflammation. J Clin Invest. 2010; 120(8):2699-2714. PubMedhttps://doi.org/10.1172/JCI39506Google Scholar
- Doedens AL, Phan AT, Stradner MH. Hypoxia-inducible factors enhance the effector responses of CD8(+) T cells to persistent antigen. Nat Immunol. 2013; 14(11):1173-1182. PubMedhttps://doi.org/10.1038/ni.2714Google Scholar
- Blouin CC, Page EL, Soucy GM, Richard DE. Hypoxic gene activation by lipopolysaccharide in macrophages: implication of hypoxia-inducible factor 1alpha. Blood. 2004; 103(3):1124-1130. PubMedhttps://doi.org/10.1182/blood-2003-07-2427Google Scholar
- Albina JE, Mastrofrancesco B, Vessella JA, Louis CA, Henry WL, Reichner JS. HIF-1 expression in healing wounds: HIF-1alpha induction in primary inflammatory cells by TNF-alpha. Am J Physiol Cell Physiol. 2001; 281(6):C1971-1977. PubMedGoogle Scholar
- Sumegi J, Barnes MG, Nestheide SV. Gene expression profiling of peripheral blood mononuclear cells from children with active hemophagocytic lymphohistiocytosis. Blood. 2011; 117(15):e151-160. PubMedhttps://doi.org/10.1182/blood-2010-08-300046Google Scholar
- Bridges JP, Lin S, Ikegami M, Shannon JM. Conditional hypoxia inducible factor-1alpha induction in embryonic pulmonary epithelium impairs maturation and augments lymphangiogenesis. Dev Biol. 2012; 362(1):24-41. PubMedhttps://doi.org/10.1016/j.ydbio.2011.10.033Google Scholar
- Narni-Mancinelli E, Chaix J, Fenis A. Fate mapping analysis of lymphoid cells expressing the NKp46 cell surface receptor. Proc Natl Acad Sci USA. 2011; 108(45):18324-18329. PubMedhttps://doi.org/10.1073/pnas.1112064108Google Scholar
- Behrens EM, Canna SW, Slade K. Repeated TLR9 stimulation results in macrophage activation syndrome-like disease in mice. J Clin Invest. 2011; 121(6):2264-2277. PubMedhttps://doi.org/10.1172/JCI43157Google Scholar
- Das R, Guan P, Sprague L. Janus kinase inhibition lessens inflammation and ameliorates disease in murine models of hemophagocytic lymphohistiocytosis. Blood. 2016; 127(13):1666-1675. PubMedhttps://doi.org/10.1182/blood-2015-12-684399Google Scholar
- Grom AA, Villanueva J, Lee S, Goldmuntz EA, Passo MH, Filipovich A. Natural killer cell dysfunction in patients with systemic-onset juvenile rheumatoid arthritis and macrophage activation syndrome. J Pediatr. 2003; 142(3):292-296. PubMedhttps://doi.org/10.1067/mpd.2003.110Google Scholar
- Ravelli A, Grom AA, Behrens EM, Cron RQ. Macrophage activation syndrome as part of systemic juvenile idiopathic arthritis: diagnosis, genetics, pathophysiology and treatment. Genes Immun. 2012; 13(4):289-298. PubMedhttps://doi.org/10.1038/gene.2012.3Google Scholar
- Villanueva J, Lee S, Giannini EH. Natural killer cell dysfunction is a distinguishing feature of systemic onset juvenile rheumatoid arthritis and macrophage activation syndrome. Arthritis Res Ther. 2005; 7(1):R30-37. PubMedhttps://doi.org/10.1186/ar1453Google Scholar
- Emig D, Salomonis N, Baumbach J, Lengauer T, Conklin BR, Albrecht M. AltAnalyze and DomainGraph: analyzing and visualizing exon expression data. Nucleic Acids Res. 2010; 38(Web Server issue):W755-762. PubMedhttps://doi.org/10.1093/nar/gkq405Google Scholar
- Chow A, Lucas D, Hidalgo A. Bone marrow CD169+ macrophages promote the retention of hematopoietic stem and progenitor cells in the mesenchymal stem cell niche. J Exp Med. 2011; 208(2):261-271. PubMedhttps://doi.org/10.1084/jem.20101688Google Scholar
- Balsamo M, Manzini C, Pietra G. Hypoxia downregulates the expression of activating receptors involved in NK-cell-mediated target cell killing without affecting ADCC. Eur J Immunol. 2013; 43(10):2756-2764. PubMedhttps://doi.org/10.1002/eji.201343448Google Scholar
- Lin N, Simon MC. Hypoxia-inducible factors: key regulators of myeloid cells during inflammation. J Clin Invest. 2016; 126(10):3661-3671. Google Scholar
- Mellins ED, Macaubas C, Grom AA. Pathogenesis of systemic juvenile idiopathic arthritis: some answers, more questions. Nat Rev Rheumatol. 2011; 7(7):416-426. PubMedhttps://doi.org/10.1038/nrrheum.2011.68Google Scholar
- Gordan JD, Thompson CB, Simon MC. HIF and c-Myc: sibling rivals for control of cancer cell metabolism and proliferation. Cancer Cell. 2007; 12(2):108-113. PubMedhttps://doi.org/10.1016/j.ccr.2007.07.006Google Scholar
- Liu L, Lu Y, Martinez J. Proinflammatory signal suppresses proliferation and shifts macrophage metabolism from Myc-dependent to HIF1alpha-dependent. Proc Natl Acad Sci USA. 2016; 113(6):1564-1569. PubMedhttps://doi.org/10.1073/pnas.1518000113Google Scholar
- Zoller EE, Lykens JE, Terrell CE. Hemophagocytosis causes a consumptive anemia of inflammation. J Exp Med. 2011; 208(6):1203-1214. PubMedhttps://doi.org/10.1084/jem.20102538Google Scholar
- Canna SW, Costa-Reis P, Bernal WE. Brief report: alternative activation of laser-captured murine hemophagocytes. Arthritis Rheum. 2014; 66(6):1666-1671. Google Scholar
- McCoy MW, Moreland SM, Detweiler CS. Hemophagocytic macrophages in murine typhoid fever have an anti-inflammatory phenotype. Infect Immun. 2012; 80(10):3642-3649. PubMedhttps://doi.org/10.1128/IAI.00656-12Google Scholar
- Canna SW, Wrobel J, Chu N, Kreiger PA, Paessler M, Behrens EM. Interferon-gamma mediates anemia but is dispensable for fulminant toll-like receptor 9-induced macrophage activation syndrome and hemophagocytosis in mice. Arthritis Rheum. 2013; 65(7):1764-1775. PubMedhttps://doi.org/10.1002/art.37958Google Scholar
- Ohyagi H, Onai N, Sato T. Monocyte-derived dendritic cells perform hemophagocytosis to fine-tune excessive immune responses. Immunity. 2013; 39(3):584-598. PubMedhttps://doi.org/10.1016/j.immuni.2013.06.019Google Scholar
- Avau A, Mitera T, Put S. Systemic juvenile idiopathic arthritis-like syndrome in mice following stimulation of the immune system with Freund’s complete adjuvant: regulation by interferon-gamma. Arthritis Rheumatol. 2014; 66(5):1340-1351. Google Scholar
- Lin FC, Karwan M, Saleh B. IFN-gamma causes aplastic anemia by altering hematopoietic stem/progenitor cell composition and disrupting lineage differentiation. Blood. 2014; 124(25):3699-3708. PubMedhttps://doi.org/10.1182/blood-2014-01-549527Google Scholar