Sustained elevation of serum interleukin-18 and its association with hemophagocytic lymphohistiocytosis in XIAP deficiency

X-linked lymphoproliferative syndrome (XLP) is a rare primary immunodeficiency characterized by increased vulnerability to Epstein–Barr virus infection. XLP type 1 is caused by mutations in SH2D1A, whereas X-linked inhibitor of apoptosis (XIAP) encoded by XIAP/BIRC4 is mutated in XLP type 2. In XIAP deficiency, hemophagocytic lymphohistiocytosis (HLH) occurs more frequently and recurrence is common. However, the underlying mechanisms remain mostly unknown. We describe the characteristics of the cytokine profiles of serum samples from 10 XIAP-deficient patients. The concentration of interleukin (IL)-18 was strikingly elevated in the patients presented with HLH, and remained high after the recovery from HLH although levels of other pro-inflammatory cytokines approached the normal range. Longitudinal examination of two patients demonstrated marked exacerbation of IL-18 levels during every occasion of HLH. These findings may suggest the association between HLH susceptibility and high serum IL-18 levels in XIAP deficiency.     

A network-informed analysis of SARS-CoV-2 and hemophagocytic lymphohistiocytosis genes’ interactions points to Neutrophil extracellular traps as mediators of thrombosis in COVID-19

Abnormal coagulation and an increased risk of thrombosis are features of severe COVID-19, with parallels proposed with hemophagocytic lymphohistiocytosis (HLH), a life-threating condition associated with hyperinflammation. The presence of HLH was described in severely ill patients during the H1N1 influenza epidemic, presenting with pulmonary vascular thrombosis. We tested the hypothesis that genes causing primary HLH regulate pathways linking pulmonary thromboembolism to the presence of SARS-CoV-2 using novel network-informed computational algorithms. This approach led to the identification of Neutrophils Extracellular Traps (NETs) as plausible mediators of vascular thrombosis in severe COVID-19 in children and adults. Taken together, the network-informed analysis led us to propose the following model: the release of NETs in response to inflammatory signals acting in concert with SARS-CoV-2 damage the endothelium and direct platelet-activation promoting abnormal coagulation leading to serious complications of COVID-19. The underlying hypothesis is that genetic and/or environmental conditions that favor the release of NETs may predispose individuals to thrombotic complications of COVID-19 due to an increase risk of abnormal coagulation. This would be a common pathogenic mechanism in conditions including autoimmune/infectious diseases, hematologic and metabolic disorders.     

Gender-associated differences of perforin polymorphisms in the susceptibility to multiple sclerosis

The granule-dependent exocytosis pathway is an important mechanism to induce apoptosis by CD8(+) T cells and NK cells and involves lytic molecules such as perforin. In the current study, we investigated the perforin 1 gene (PRF1) as a candidate for multiple sclerosis (MS) susceptibility in the Spanish population. We genotyped three PRF1 single nucleotide polymorphisms (rs885822, rs10999426, and rs3758562) in 420 patients with MS and 512 controls. Associations of PRF1 polymorphisms with the disease were restricted to male patients with MS, and the finding was consistently observed at the allele, genotype, and haplotype levels. Gender-associated differences were validated in an additional replication cohort comprised of 292 MS cases and 300 controls. In addition, we identified minor risk haplotypes strongly associated with male patients having primary progressive MS (PPMS). Further characterization of male patients with PPMS carrying the risk haplotypes by means of gene expression microarrays revealed overrepresentation of the cell killing gene ontology category among downregulated genes in these patients compared with male patients with PPMS carrying protective haplotypes. Moreover, PRF1 mRNA expression levels were significantly lower in patients with risk haplotypes, and changes in perforin protein expression by CD8(+) T cells mirrored those observed in gene expression. These findings suggest a gender dimorphism in the PRF1 association with MS and point to the presence of a generalized defect in the expression of genes that code for proteins involved in cell killing in a subgroup of male patients with PPMS.     

Tumor necrosis factor alpha promoter polymorphism associated with increased susceptibility to secondary hemophagocytic lymphohistiocytosis in the Korean population

Secondary hemophagocytic lymphohistiocytosis (HLH) can be associated with various diseases including infections and lymphoma. The clinical findings of HLH can be explained by an increased production of cytokines such as tumor necrosis factor alpha (TNF-alpha). As not all the patients with infection or lymphoma have secondary HLH, we investigated the relationship between susceptibility to secondary HLH and TNF-alpha promoter polymorphisms to identify genetic factors of secondary HLH. We determined the alleles of four promoter sites (-1031/-857/-308/-238) of TNF-alpha gene by using Taqman-based allelic discrimination assays in the 66 Korean patients with secondary HLH and 100 healthy Korean controls. We found that the frequency of the TNF-alpha -1031C allele, which is associated with higher-plasma TNF-alpha levels, was enriched in patients with secondary HLH compared with healthy controls (OR=2.00, 95% CI 1.20-3.30, P=0.007). In haplotype analysis of TNF-alpha polymorphisms, the haplotype H6 (CTGG) was detected only in the patient group, and the haplotype group (H2 or H5 or H6) including TNF-alpha -1031C allele was overexpressed in secondary HLH patients (OR=2.52, 95% CI 1.33-4.77, P=0.004). These results suggested that TNF-alpha -1031C allele and its associated haplotypes in Koreans may enhance susceptibility to secondary HLH.     

Integration of FGF and TWIST in calvarial bone and suture development

Mutations in the FGFR1-FGFR3 and TWIST genes are known to cause craniosynostosis, the former by constitutive activation and the latter by haploinsufficiency. Although clinically achieving the same end result, the premature fusion of the calvarial bones, it is not known whether these genes lie in the same or independent pathways during calvarial bone development and later in suture closure. We have previously shown that Fgfr2c is expressed at the osteogenic fronts of the developing calvarial bones and that, when FGF is applied via beads to the osteogenic fronts, suture closure is accelerated (Kim, H.-J., Rice, D. P. C., Kettunen, P. J. and Thesleff, I. (1998) Development 125, 1241-1251). In order to investigate further the role of FGF signalling during mouse calvarial bone and suture development, we have performed detailed expression analysis of the splicing variants of Fgfr1-Fgfr3 and Fgfr4, as well as their potential ligand Fgf2. The IIIc splice variants of Fgfr1-Fgfr3 as well as the IIIb variant of Fgfr2 being expressed by differentiating osteoblasts at the osteogenic fronts (E15). In comparison to Fgf9, Fgf2 showed a more restricted expression pattern being primarily expressed in the sutural mesenchyme between the osteogenic fronts. We also carried out a detailed expression analysis of the helix-loop-helix factors (HLH) Twist and Id1 during calvaria and suture development (E10-P6). Twist and Id1 were expressed by early preosteoblasts, in patterns that overlapped those of the FGF ligands, but as these cells differentiated their expression dramatically decreased. Signalling pathways were further studied in vitro, in E15 mouse calvarial explants. Beads soaked in FGF2 induced Twist and inhibited Bsp, a marker of functioning osteoblasts. Meanwhile, BMP2 upregulated Id1. Id1 is a dominant negative HLH thought to inhibit basic HLH such as Twist. In Drosophila, the FGF receptor FR1 is known to be downstream of Twist. We demonstrated that in Twist(+/)(-) mice, FGFR2 protein expression was altered. We propose a model of osteoblast differentiation integrating Twist and FGF in the same pathway, in which FGF acts both at early and late stages. Disruption of this pathway may lead to craniosynostosis.     

Secondary hemophagocytic lymphohistiocytosis versus cytokine release syndrome in severe COVID-19 patients

COVID-19 or SARS-CoV-2 infection can lead to severe acute respiratory distress syndrome/pneumonia with features of cytokine storm reminiscent of secondary hemophagocytic lymphohistiocytosis (HLH), which can be diagnosed by the calculated HScore. Recent reports have suggested favorable responses to the interleukin-1 receptor antagonist, anakinra in patients with COVID-19 associated secondary HLH. In our single institution study, we compared 14 COVID-19 cytokine storm patients with 10 secondary HLH patients seen immediately prior to the pandemic (non-COVID-19), to determine whether diagnostic features of secondary HLH were typically seen in COVID-19 patients presenting with cytokine storm. Although most of our COVID-19 patients did not fulfill diagnostic criteria for HLH, we hypothesize that identification of HLH may relate to the severity or timing of cytokine release. Based on our observations, we would suggest distinguishing between cytokine release syndrome and secondary HLH, reserving the latter term for cases fulfilling diagnostic criteria.Impact statementSevere COVID-19 associated pneumonia and acute respiratory distress syndrome has recently been described with life-threatening features of cytokine storm and loosely referred to as hemophagocytic lymphohistiocytosis (HLH) or macrophage activation syndrome (MAS). Although a recent report indicated favorable responses to the interleukin-1 receptor antagonist, anakinra in eight patients with COVID-19 secondary HLH diagnosed using the HScore calculation, others have suggested that the diagnosis of secondary HLH is uncommon and that the use of the HScore has limited value in guiding immunomodulatory therapy for COVID-19. Here, we provide additional perspective on this important controversy based upon comparisons between 14 COVID-19 cytokine storm patients and 10 secondary HLH patients seen immediately prior to the pandemic. We hypothesize that identification of HLH may relate to the severity or timing of cytokine release and suggest distinguishing between cytokine release syndrome and secondary HLH, reserving the latter term for cases fulfilling diagnostic criteria.     

Pathophysiology and spectrum of diseases caused by defects in lymphocyte cytotoxicity

In experimental settings, lymphocyte cytotoxicity has been recognized as a central mechanism for immune defense against infected and neoplastic cells. More recently, molecular determinants of lymphocyte cytotoxicity have been identified through studies of rare, inherited hyperinflammatory and lymphoproliferative syndromes that include hemophagocytic lymphohistiocytosis (HLH). These studies have unraveled a set of genes pivotal for the biogenesis and directed release of perforin-containing lysosomes that mediate target cell killing, in addition to other pathways including Fas that also contribute to induction of cell death. Furthermore, studies of such human primary immunodeficiencies have highlighted non-redundant roles of perforin for maintenance of immune homeostasis. Besides providing mechanistic insights to lymphocyte cytotoxicity, studies of individuals with rare hyperinflammatory diseases are highlighting the relevance of lymphocyte cytotoxicity to more common human diseases. It is increasingly recognized that mutations abrogating lymphocyte cytotoxicity not only cause HLH, but also are associated with susceptibility to cancer and autoimmune syndromes. In addition, patients may initially be present with neurological symptoms or severe infectious disease masquerading as variable immunodeficiency syndrome. Here, we highlight new knowledge regarding the molecular mechanisms regulating lymphocyte cytotoxicity and review how mutations in genes associated with HLH cause disease. We also discuss the wider implications of impairments in lymphocyte cytotoxicity for human disease predisposition.     

SREBP-1 dimerization specificity maps to both the helix-loop-helix and leucine zipper domains: use of a dominant negative

The mammalian SREBP family contains two genes that code for B-HLH-ZIP proteins that bind sequence-specific DNA to regulate the expression of genes involved in lipid metabolism. We have designed a dominant negative (DN), termed A-SREBP-1, that inhibits the DNA binding of either SREBP protein. A-SREBP-1 consists of the dimerization domain of B-SREBP-1 and a polyglutamic acid sequence that replaces the basic region. A-SREBP-1 heterodimerizes with either B-SREBP-1 or B-SREBP-2, and both heterodimers are more stable than B-SREBP-1 bound to DNA. Circular dichroism thermal denaturation studies show that the B-SREBP-1.A-SREBP-1 heterodimer is -9.8 kcal mol(-1) dimer(-1) more stable than the B-SREBP-1 homodimer. EMSA assays demonstrate that A-SREBP-1 can inhibit the DNA binding of either B-SREBP-1 or B-SREBP-2 in an equimolar competition but does not inhibit the DNA binding of the three B-HLH-ZIP proteins MAX, USF, or MITF, even at 100 molar eq. Chimeric proteins containing the HLH domain of SREBP-1 and the leucine zipper from either MAX, USF, or MITF indicate that both the HLH and leucine zipper regions of SREBP-1 contribute to its dimerization specificity. Transient co-transfection studies demonstrate that A-SREBP-1 can inhibit the transactivation of SREBP-1 and SREBP-2 but not USF. A-SREBP-1 may be useful in metabolic diseases where SREBP family members are overexpressed.     

IDO1 Deficiency Does Not Affect Disease in Mouse Models of Systemic Juvenile Idiopathic Arthritis and Secondary Hemophagocytic Lymphohistiocytosis

Objectives

Indoleamine 2,3-dioxygenase-1 (IDO1) is an immune-modulatory enzyme that catalyzes the degradation of tryptophan (Trp) to kynurenine (Kyn) and is strongly induced by interferon (IFN)-γ. We previously reported highly increased levels of IFN-γ and corresponding IDO activity in patients with hemophagocytic lymphohistiocytosis (HLH), a hyper-inflammatory syndrome. On the other hand, IFN-γ and IDO were low in patients with systemic juvenile idiopathic arthritis (sJIA), an autoinflammatory syndrome. As HLH can occur as a complication of sJIA, the opposing levels of both IFN-γ and IDO are remarkable. In animal models for sJIA and HLH, the role of IFN-γ differs from being protective to pathogenic. In this study, we aimed to unravel the role of IDO1 in the pathogenesis of sJIA and HLH.

Methods

Wild-type and IDO1-knockout (IDO1-KO) mice were used in 3 models of sJIA or HLH: complete Freund’s adjuvant (CFA)-injected mice developed an sJIA-like syndrome and secondary HLH (sHLH) was evoked by either repeated injection of unmethylated CpG oligonucleotide or by primary infection with mouse cytomegalovirus (MCMV). An anti-CD3-induced cytokine release syndrome was used as a non-sJIA/HLH control model.

Results

No differences were found in clinical, laboratory and hematological features of sJIA/HLH between wild-type and IDO1-KO mice. As IDO modulates the immune response via induction of regulatory T cells and inhibition of T cell proliferation, we investigated both features in a T cell-triggered cytokine release syndrome. Again, no differences were observed in serum cytokine levels, percentages of regulatory T cells, nor of proliferating or apoptotic thymocytes and lymph node cells.

Conclusions

Our data demonstrate that IDO1 deficiency does not affect inflammation in sJIA, sHLH and a T cell-triggered cytokine release model. We hypothesize that other tryptophan-catabolizing enzymes like IDO2 and tryptophan 2,3-dioxygenase (TDO) might compensate for the lack of IDO1.     

Immunologic Difference between Hypersensitivity to Mosquito Bite and Hemophagocytic Lymphohistiocytosis Associated with Epstein-Barr Virus Infection

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening, virus-triggered immune disease. Hypersensitivity to mosquito bite (HMB), a presentation of Chronic Active Epstein-Barr Virus infection (CAEBV), may progress to HLH. This study aimed to investigate the immunologic difference between the HMB episodes and the HLH episodes associated with EBV infection. Immunologic changes of immunoglobulins, lymphocyte subsets, cytotoxicity, intracellular perforin and granzyme expressions, EBV virus load and known candidate genes for hereditary HLH were evaluated and compared. In 12 HLH episodes (12 patients) and 14 HMB episodes (4 patients), there were both decreased percentages of CD4+ and CD8+ and increased memory CD4+ and activated (CD2+HLADR+) lymphocytes. In contrast to HMB episodes that had higher IgE levels and EBV virus load predominantly in NK cells, those HLH episodes with virus load predominantly in CD3+ lymphocyte had decreased perforin expression and cytotoxicity that were recovered in the convalescence period. However, there was neither significant difference of total virus load in these episodes nor candidate genetic mutations responsible for hereditary HLH. In conclusion, decreased perforin expression in the HLH episodes with predominant-CD3+ EBV virus load is distinct from those HMB episodes with predominant-NK EBV virus load. Whether the presence of non-elevated memory CD4+ cells or activated lymphocytes (CD2+HLADR+) increases the mortality rate in the HLH episodes remains to be further warranted through larger-scale studies.     

Is COVID-19-associated cytokine storm distinct from non-COVID-19 secondary hemophagocytic lymphohistiocytosis?

Cytokine storm is an umbrella term that describes an inflammatory syndrome characterized by elevated levels of circulating cytokines and hyperactivation of innate and/or adaptive immune cells. One type of cytokine storm is hemophagocytic lymphohistiocytosis (HLH), which can be either primary or secondary. Severe COVID-19-associated pneumonia and acute respiratory distress syndrome (ARDS) can also lead to cytokine storm/cytokine release syndrome (CS/CRS) and, more rarely, meet criteria for the diagnosis of secondary HLH. Here, we review the immunobiology of primary and secondary HLH and examine whether COVID-19-associated CS/CRS can be discriminated from non-COVID-19 secondary HLH. Finally, we review differences in immunobiology between these different entities, which may inform both clinical diagnosis and treatment of patients.     

Pulsed Radiofrequency Reduced Complete Freund’s Adjuvant-induced Mechanical Hyperalgesia via the Spinal c-Jun N-terminal Kinase Pathway

Pulsed radiofrequency (PRF) treatment involves the pulsed application of a radiofrequency electric field to a nerve. The technology offers pain relief for patients suffering from chronic pain who do not respond well to conventional treatments. We tested whether PRF treatment attenuated complete Freund’s adjuvant (CFA) induced inflammatory pain. The profile of spinal c-Jun N-terminal kinases (JNKs) phosphorylation was evaluated to elucidate the potential mechanism. Injection of CFA into the unilateral hind paw of rats induced mechanical hyperalgesia in both the ipsilateral and contralateral hind paws. We administered 500-kHz PRF treatment in 20-ms pulses, at a rate of 2 Hz (2 pulses per second) either to the sciatic nerve in the mid-thigh, or to the L4 anterior primary ramus just distal to the intervertebral foramen in both the CFA group and no-PRF group rats. Tissue samples were examined at 1, 3, 7, and 14 days following PRF treatments. Behavioral studies showed that PRF applied close to the dorsal root ganglion (DRG) significantly attenuated CFA-induced mechanical hyperalgesia compared to no-PRF group (P < .05). And western blotting revealed significant attenuation of the activation of JNK in the spinal dorsal horn compared to no-PRF group animals (P < .05). Application of PRF close to DRG provides an effective treatment for CFA-induced persistent mechanical hyperalgesia by attenuating JNK activation in the spinal dorsal horn.     

Distinct roles of the first introns on the expression of Arabidopsis profilin gene family members

Profilin is a small actin-binding protein that regulates cellular dynamics of the actin cytoskeleton. In Arabidopsis (Arabidopsis thaliana), five profilins were identified. The vegetative class profilins, PRF1, PRF2, and PRF3, are expressed in vegetative organs. The reproductive class profilins, PRF4 and PRF5, are mainly expressed in pollen. In this study, we examined the role of the first intron in the expression of the Arabidopsis profilin gene family using transgenic plants and a transient expression system. In transgenic plants, we examined PRF2 and PRF5, which represent vegetative and reproductive profilins. The expression of the PRF2 promoter fused with the beta-glucuronidase (GUS) gene was observed in the vascular bundles, but transgenic plants carrying the PRF2 promoter-GUS with its first intron showed constitutive expression throughout the vegetative tissues. However, the first intron of PRF5 had little effect on the reporter gene expression pattern. Transgenic plants containing PRF5 promoter-GUS fusion with or without its first intron showed reproductive tissue-specific expression. To further investigate the different roles of the first two introns on gene expression, the first introns were exchanged between PRF2 and PRF5. The first intron of PRF5 had no apparent effect on the expression pattern of the PRF2 promoter. But, unlike the intron of PRF5, the first intron of PRF2 greatly affected the reproductive tissue-specific expression of the PRF5 promoter, confirming a different role for these introns. The results of a transient expression assay indicated that the first intron of PRF1 and PRF2 enhances gene expression, whereas PRF4 and PRF5 do not. These results suggest that the first introns of profilin genes are functionally distinctive and the first introns are required for the strong and constitutive gene expression of PRF1 and PRF2 in vegetative tissues.     

Subtypes of familial hemophagocytic lymphohistiocytosis in Japan based on genetic and functional analyses of cytotoxic T lymphocytes

Background

Familial hemophagocytic lymphohistiocytosis (FHL) is a rare disease of infancy or early childhood. To clarify the incidence and subtypes of FHL in Japan, we performed genetic and functional analyses of cytotoxic T lymphocytes (CTLs) in Japanese patients with FHL.

Design and Methods

Among the Japanese children with hemophagocytic lymphohistiocytosis (HLH) registered at our laboratory, those with more than one of the following findings were eligible for study entry under a diagnosis of FHL: positive for known genetic mutations, a family history of HLH, and impaired CTL-mediated cytotoxicity. Mutations of the newly identified causative gene for FHL5, STXBP2, and the cytotoxicity and degranulation activity of CTLs in FHL patients, were analyzed.

Results

Among 31 FHL patients who satisfied the above criteria, PRF1 mutation was detected in 17 (FHL2) and UNC13D mutation was in 10 (FHL3). In 2 other patients, 3 novel mutations of STXBP2 gene were confirmed (FHL5). Finally, the remaining 2 were classified as having FHL with unknown genetic mutations. In all FHL patients, CTL-mediated cytotoxicity was low or deficient, and degranulation activity was also low or absent except FHL2 patients. In 2 patients with unknown genetic mutations, the cytotoxicity and degranulation activity of CTLs appeared to be deficient in one patient and moderately impaired in the other.

Conclusions

FHL can be diagnosed and classified on the basis of CTL-mediated cytotoxicity, degranulation activity, and genetic analysis. Based on the data obtained from functional analysis of CTLs, other unknown gene(s) responsible for FHL remain to be identified.     

Clinical and laboratory characteristics of hemophagocytic lymphohistiocytosis induced by Leishmania infantum infection

BackgroundVisceral leishmaniasis (VL) could progress to secondary hemophagocytic lymphohistiocytosis (HLH), which is a rare but life-threatening condition with poor prognosis. So far, the clinical and laboratory characteristics of VL associated HLH have not been well elucidated.Method and findingsIn this study, we retrospectively analyzed the clinical and laboratory profiles between 17 patients with VL associated HLH and 27 patients with VL alone admitted at the Beijing Friendship Hospital, Capital Medical University from May 2016 to March 2021. In addition to the identification of Leishmania infection, hemophagocytosis was identified in bone marrow in the most cases of VL associated HLH (15/17). The patients with VL associated HLH had higher chances of bleeding, hepatomegaly, thrombocytopenia, hypertriglyceridemia, hyperferritinemia, hypofibrinogenemia, elevated secretion of soluble IL-2 receptor or lower NK cell activity compared to patients with VL only. Furthermore, patients with VL associated HLH had higher inflammation status associated with higher levels of Th1 (TNF-α, IFN-γ, IL-1beta, IL-6, IL-8, IL-12p70), Th2 (IL-4) and Th17 cytokines (IL-17, IL-23) in the peripheral blood, and higher parasite load (qPCR and parasite culture). All 27 VL cases were totally recovered after being treated with Sodium Stibogluconate, five of the 17 patients with VL associated HLH died even after timely treatment with anti-parasite and immunosuppressive chemotherapy.ConclusionWithout appropriate treatment, visceral leishmaniosis could develop to secondary HLH. The parasite culturing and qPCR detection of bone marrow samples facilitates the diagnosis of VL associated HLH in addition to other findings of HLH. Prompt treatment with anti-Leishmania and immunosuppressive chemotherapy is critical to reduce the mortality of VL associated HLH.