Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hematologic disorder caused by uncontrolled proliferation of activated diverse immune cells. Primary HLH, also known as familial hemophagocytic lymphohistiocytosis (FHL), is a heterogeneous autosomal recessive disorder found to be more prevalent with parental consanguinity. Secondary HLH occurs after strong immunologic activation, such as that which can occur with systemic infection, immunodeficiency, or underlying malignancy. Despite the fact that different genetic/somatic aberrations and different NK cell activities have been reported between primary and secondary HLH, the key features for HLH (cytokine storm, fever, hepatosplenomegaly, lymphadenopathy, jaundice and a rash) remain common.

To identify the key pathobiologic mediators for HLH, we first analyzed two published microarray expression data sets of peripheral blood mononuclear cells from the patients with FHL and systemic juvenile idiopathic arthritis (sJIA), which is closely associated with macrophage activation syndrome, a subtype of secondary HLH. The FHL cohort contains 33 healthy donors and 11 FHL patients, and the sJIA cohort contains 17 sJIA patients and 30 healthy donors. TF-target enrichment analysis revealed that HIF1A is one of the top predicted key transcription factors regulating up-regulated genes in the both cohorts. To determine the HIF1A signature in HLH, we then performed gene set enrichment analysis (GSEA) using these two data sets. GSEA revealed that the HIF1A-induced signature in cord blood CD34+ cells is significantly enriched in the both data sets. These results further suggest that HIF1A may play a key role in regulating downstream targets in both primary and secondary HLH patients.

We determined whether HIF1A signaling is activated in the established HLH mouse models. The LCMV-infected perforin-knockout (Prf1-/-) mouse model is a primary HLH mouse model. Repeated injections of TLR9 ligand CpG oligodeoxynucleotides result in secondary HLH in wild-type (WT) mice. Those models quickly develop HLH phenotypes after LCMV challenge and CpG injections, respectively. The expression levels of HIF1A protein in the spleen macrophages from both LCMV-infected Prf1-/- mice and CpG-treated mice were significantly elevated compared to the control mice. Bone marrow and spleen macrophages were polarized to type 1 phenotype in the both models.

To determine the sufficient role of the HIF1A signaling activation in developing HLH phenotype, we combined the Vav1-Cre allele, Rosa26-loxp-stop-loxp (LSL) reverse-tetracycline-controlled transactivator (rtTA) allele, and HIF1A-triple point mutant (TPM); and generated transgenic mice with blood specific inducible HIF1A protein expression (Vav1-Cre/TPM mice). The expression of HIF1A protein in Vav1-Cre/TPM mice was elevated in both hematopoietic stem and progenitor cells and mature cells compared to the control mice. In the pure B6 background, these mice developed HLH phenotypes within 3 weeks after doxycycline administration, such as pancytopenia, bone marrow failure, liver damage, ferritinemia, and splenomegaly. The expression of serum inflammatory cytokines, such as IL-6, IL-12, IFN-γ, and GM-CSF was increased in the Vav1-Cre/TPM mice. Type 1 polarized macrophages and dysregulated NK cells were found in bone marrow and spleen from the Vav1-Cre/TPM mice.

Since an activation of cytotoxic T cells and IFN-γ signaling play a critical role in the LCMV-infected Prf1-/- primary HLH mouse model and type 1 macrophage polarization, we evaluated the significance of adaptive immunity by using recombination activation gene 1 (Rag1)-deficient (Rag1-/-) mice (which lack T cells and B cells) and Ifng-knockout (Ifng-/-) mice. Rag1-/-/ Vav1 -Cre/TPM mice developed similar phenotypes as the Vav1 -Cre/TPM mice. Type 1 macrophage polarization was also observed in both mice. These results indicate that activation of adaptive immunity and IFN-γ signaling is the upstream of HIF1A signaling. To confirm this, we treated Raw264.7 cells, a murine macrophage cell line, with IFN-γ, and found both activation of several HIF1A target gene expression and the polarization toward type 1 phenotype.

In conclusion, our data reveals that HIF1A signaling is a critical downstream mediator for HLH and could be a novel therapeutic target for HLH.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.

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