Advances in Immune Tolerance Induction in Enzyme Replacement Therapy.

Inborn errors of metabolism (IEMs) are a group of genetic diseases that occur due to the either deficiency of an enzyme involved in a metabolic/biochemical pathway or other disturbances in the metabolic pathway including transport protein or activator protein deficiencies, cofactor deficiencies, organelle biogenesis, maturation or trafficking problems. These disorders are collectively significant due to their substantial impact on both the well-being and survival of affected individuals. In the quest for effective treatments, enzyme replacement therapy (ERT) has emerged as a viable strategy for patients with many of the lysosomal storage disorders (LSD) and enzyme substitution therapy in the rare form of the other inborn errors of metabolism including phenylketonuria and hypophosphatasia. However, a major challenge associated with enzyme infusion in patients with these disorders, mainly LSD, is the development of high antibody titres. Strategies focusing on immunomodulation have shown promise in inducing immune tolerance to ERT, leading to improved overall survival rates. The implementation of immunomodulation concurrent with ERT administration has also resulted in a decreased occurrence of IgG antibody development compared with cases treated solely with ERT. By incorporating the knowledge gained from current approaches and analysing the outcomes of immune tolerance induction (ITI) modalities from clinical and preclinical trials have demonstrated significant improvement in the efficacy of ERT. In this comprehensive review, the progress in ITI modalities is assessed, drawing insights from both clinical and preclinical trials. The focus is on evaluating the advancements in ITI within the context of IEM, specifically addressing LSDs managed through ERT.

Cellular mechanisms and clinical applications for phenocopies of inborn errors of immunity: infectious susceptibility due to cytokine autoantibodies.

INTRODUCTION: With a growing knowledge of Inborn errors immunity (IEI), immunological profiling and genetic predisposition to IEI phenocopies have been developed in recent years. AREAS COVERED: Here we summarized the correlation between various pathogen invasions, autoantibody profiles, and corresponding clinical features in the context of patients with IEI phenocopies. It has been extensively evident that patients with anti-cytokine autoantibodies underly impaired anti-pathogen immune responses and lead to broad unregulated inflammation and tissue damage. Several hypotheses of anti-cytokine autoantibodies production are summarized here, including a defective negative selection of autoreactive T cells, abnormal germinal center formation, molecular mimicry, HLA class II allele region, lack of auto-reactive lymphocyte apoptosis, and other possible hypotheses. EXPERT OPINION: Phenocopies of IEI associated with anti-cytokine autoantibodies are increasingly recognized as one of the causes of acquired immunodeficiency and susceptibility to certain pathogen infections, especially facing the current challenge of the COVID-19 pandemic. By investigating clinical, genetic, and pathogenesis autoantibodies profiles associated with various pathogens' susceptibilities, we could better understand the IEI phenocopies with anti-cytokine autoantibodies, especially for those that underlie life-threatening SARS-CoV-2.

Inborn errors of TLR3- or MDA5-dependent type I IFN immunity in children with enterovirus rhombencephalitis.

Enterovirus (EV) infection rarely results in life-threatening infection of the central nervous system. We report two unrelated children with EV30 and EV71 rhombencephalitis. One patient carries compound heterozygous TLR3 variants (loss-of-function F322fs2* and hypomorphic D280N), and the other is homozygous for an IFIH1 variant (loss-of-function c.1641+1G>C). Their fibroblasts respond poorly to extracellular (TLR3) or intracellular (MDA5) poly(I:C) stimulation. The baseline (TLR3) and EV-responsive (MDA5) levels of IFN-ß in the patients' fibroblasts are low. EV growth is enhanced at early and late time points of infection in TLR3- and MDA5-deficient fibroblasts, respectively. Treatment with exogenous IFN-α2b before infection renders both cell lines resistant to EV30 and EV71, whereas post-infection treatment with IFN-α2b rescues viral susceptibility fully only in MDA5-deficient fibroblasts. Finally, the poly(I:C) and viral phenotypes of fibroblasts are rescued by the expression of WT TLR3 or MDA5. Human TLR3 and MDA5 are critical for cell-intrinsic immunity to EV, via the control of baseline and virus-induced type I IFN production, respectively.

Infection-induced inflammation from specific inborn errors of immunity to COVID-19.

Inborn errors of immunity (IEIs) are a group of genetically defined disorders leading to defective immunity. Some IEIs have been linked to mutations of immune receptors or signaling molecules, resulting in defective signaling of respective cascades essential for combating specific pathogens. However, it remains incompletely understood why in selected IEIs, such as X-linked lymphoproliferative syndrome type 2 (XLP-2), hypo-immune response to specific pathogens results in persistent inflammation. Moreover, mechanisms underlying the generation of anticytokine autoantibodies are mostly unknown. Recently, IEIs have been associated with coronavirus disease 2019 (COVID-19), with a small proportion of patients that contract severe COVID-19 displaying loss-of-function mutations in genes associated with type I interferons (IFNs). Moreover, approximately 10% of patients with severe COVID-19 possess anti-type I IFN-neutralizing autoantibodies. Apart from IEIs that impair immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV-2 encodes several proteins that suppress early type I IFN production. One primary consequence of the lack of type I IFNs during early SARS-CoV-2 infection is the increased inflammation associated with COVID-19. In XLP-2, resolution of inflammation rescued experimental subjects from infection-induced mortality. Recent studies also indicate that targeting inflammation could alleviate COVID-19. In this review, we discuss infection-induced inflammation in IEIs, using XLP-2 and COVID-19 as examples. We suggest that resolving inflammation may represent an effective therapeutic approach to these diseases.

ß3-Adrenoreceptors as ROS Balancer in Hematopoietic Stem Cell Transplantation.

In the last decades, the therapeutic potential of hematopoietic stem cell transplantation (HSCT) has acquired a primary role in the management of a broad spectrum of diseases including cancer, hematologic conditions, immune system dysregulations, and inborn errors of metabolism. The different types of HSCT, autologous and allogeneic, include risks of severe complications including acute and chronic graft-versus-host disease (GvHD) complications, hepatic veno-occlusive disease, lung injury, and infections. Despite being a dangerous procedure, it improved patient survival. Hence, its use was extended to treat autoimmune diseases, metabolic disorders, malignant infantile disorders, and hereditary skeletal dysplasia. HSCT is performed to restore or treat various congenital conditions in which immunologic functions are compromised, for instance, by chemo- and radiotherapy, and involves the administration of hematopoietic stem cells (HSCs) in patients with depleted or dysfunctional bone marrow (BM). Since HSCs biology is tightly regulated by oxidative stress (OS), the control of reactive oxygen species (ROS) levels is important to maintain their self-renewal capacity. In quiescent HSCs, low ROS levels are essential for stemness maintenance; however, physiological ROS levels promote HSC proliferation and differentiation. High ROS levels are mainly involved in short-term repopulation, whereas low ROS levels are associated with long-term repopulating ability. In this review, we aim summarize the current state of knowledge about the role of ß3-adrenoreceptors (ß3-ARs) in regulating HSCs redox homeostasis. ß3-ARs play a major role in regulating stromal cell differentiation, and the antagonist SR59230A promotes differentiation of different progenitor cells in hematopoietic tumors, suggesting that ß3-ARs agonism and antagonism could be exploited for clinical benefit.

Inborn errors of immunity and metabolic disorders: current understanding, diagnosis, and treatment approaches.

Inborn errors of metabolism consist of a heterogeneous group of disorders with various organ systems manifestations, and some metabolic diseases also cause immunological disorders or dysregulation. In this review, metabolic diseases that affect the immunological system and particularly lead to primary immune deficiency will be reviewed. In a patient with frequent infections and immunodeficiency, the presence of symptoms such as growth retardation, abnormal facial appearance, heart, skeletal, lung deformities, skin findings, arthritis, motor developmental retardation, seizure, deafness, hepatomegaly, splenomegaly, impairment of liver function tests, the presence of anemia, thrombocytopenia and eosinophilia in hematological examinations should suggest metabolic diseases for the underlying cause. In some patients, these phenotypic findings may appear before the immunodeficiency picture. Metabolic diseases leading to immunological disorders are likely to be rare but probably underdiagnosed. Therefore, the presence of recurrent infections or autoimmune findings in a patient with a suspected metabolic disease should suggest that immune deficiency may also accompany the picture, and diagnostic examinations in this regard should be deepened.

Negative selection on human genes underlying inborn errors depends on disease outcome and both the mode and mechanism of inheritance.

Genetic variants underlying life-threatening diseases, being unlikely to be transmitted to the next generation, are gradually and selectively eliminated from the population through negative selection. We study the determinants of this evolutionary process in human genes underlying monogenic diseases by comparing various negative selection scores and an integrative approach, CoNeS, at 366 loci underlying inborn errors of immunity (IEI). We find that genes underlying autosomal dominant (AD) or X-linked IEI have stronger negative selection scores than those underlying autosomal recessive (AR) IEI, whose scores are not different from those of genes not known to be disease causing. Nevertheless, genes underlying AR IEI that are lethal before reproductive maturity with complete penetrance have stronger negative selection scores than other genes underlying AR IEI. We also show that genes underlying AD IEI by loss of function have stronger negative selection scores than genes underlying AD IEI by gain of function, while genes underlying AD IEI by haploinsufficiency are under stronger negative selection than other genes underlying AD IEI. These results are replicated in 1,140 genes underlying inborn errors of neurodevelopment. Finally, we propose a supervised classifier, SCoNeS, which predicts better than state-of-the-art approaches whether a gene is more likely to underlie an AD or AR disease. The clinical outcomes of monogenic inborn errors, together with their mode and mechanisms of inheritance, determine the levels of negative selection at their corresponding loci. Integrating scores of negative selection may facilitate the prioritization of candidate genes and variants in patients suspected to carry an inborn error.

Stem Cell-Based Disease Models for Inborn Errors of Immunity.

The intrinsic capacity of human hematopoietic stem cells (hHSCs) to reconstitute myeloid and lymphoid lineages combined with their self-renewal capacity hold enormous promises for gene therapy as a viable treatment option for a number of immune-mediated diseases, most prominently for inborn errors of immunity (IEI). The current development of such therapies relies on disease models, both in vitro and in vivo, which allow the study of human pathophysiology in great detail. Here, we discuss the current challenges with regards to developmental origin, heterogeneity and the subsequent implications for disease modeling. We review models based on induced pluripotent stem cell technology and those relaying on use of adult hHSCs. We critically review the advantages and limitations of current models for IEI both in vitro and in vivo. We conclude that existing and future stem cell-based models are necessary tools for developing next generation therapies for IEI.

Unexpected combination: DiGeorge syndrome and myeloperoxidase deficiency.

We report a case of a 3-year-old boy who presented with recurrent bacterial and fungal infections and a known diagnosis of partial DiGeorge (22q11.2 deletion) syndrome. The nature and severity of his infections were more than normally expected in partial DiGeorge syndrome with normal T-cell counts and T-cell proliferative response to phytohaemagglutinin. This prompted further investigation of the immune system. An abnormal neutrophil respiratory oxidative burst, but normal protein expression of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system, led to the identification of myeloperoxidase deficiency. DiGeorge syndrome has a heterogeneous clinical phenotype and may not be an isolated diagnosis. It raises awareness of the possibility of two rare diseases occurring in a single patient and emphasises that even when a rare diagnosis is confirmed, if the clinical features remain atypical or unresponsive, then further investigation for additional cofactors is warranted.

Mannan-binding lectin promotes keratinocyte to produce CXCL1 and enhances neutrophil infiltration at the early stages of psoriasis.

Psoriasis is a chronic, relapsing inflammatory skin disorder. Numerous experimental evidence and therapeutic evidence have shown that the innate immune response is critical for the pathogenesis and development of psoriasis. Mannan-binding lectin (MBL), a prototypic pattern recognition molecule of the innate immune system, plays an essential role in the host defense against certain infections and also appears to be a major regulator of inflammation. In this study, we investigated the function of MBL on the course of experimental murine imiquimod (IMQ)-induced psoriasis. Our data showed that MBL-deficient (MBL-/- ) mice exhibited attenuated skin damage characterized by greatly decreased erythema compared with wild-type control mice during the early stages of IMQ-induced psoriasis-like skin inflammation. The reduced skin inflammation in MBL-/- mice was associated with the decreased infiltration of neutrophils. Furthermore, we have determined that MBL deficiency limited the chemokine CXCL1 production from skin keratinocytes upon IMQ stimulation, which might be responsible for the impaired skin recruitment of neutrophils. Additionally, we have provided the data that MBL protein promotes the IMQ-induced expression of CXCL1 and activation of MAPK/NF-κB signalling pathway in human keratinocyte HaCaT cells in vitro. In summary, our study revealed an unexpected role of MBL on keratinocyte function in skin, thus offering a new insight into the pathogenic mechanisms of psoriasis.

A General Introduction to Glucocorticoid Biology.

Glucocorticoids (GCs) are steroid hormones widely used for the treatment of inflammation, autoimmune diseases, and cancer. To exert their broad physiological and therapeutic effects, GCs bind to the GC receptor (GR) which belongs to the nuclear receptor superfamily of transcription factors. Despite their success, GCs are hindered by the occurrence of side effects and glucocorticoid resistance (GCR). Increased knowledge on GC and GR biology together with a better understanding of the molecular mechanisms underlying the GC side effects and GCR are necessary for improved GC therapy development. We here provide a general overview on the current insights in GC biology with a focus on GC synthesis, regulation and physiology, role in inflammation inhibition, and on GR function and plasticity. Furthermore, novel and selective therapeutic strategies are proposed based on recently recognized distinct molecular mechanisms of the GR. We will explain the SEDIGRAM concept, which was launched based on our research results.

Heterogeneity of hypochlorous acid production in individual neutrophil phagosomes revealed by a rhodamine-based probe.

The rhodamine-based probe R19-S has been shown to react with hypochlorous acid (HOCl) to yield fluorescent R19, but not with some other oxidants including hydrogen peroxide. Here, we further examined the specificity of R19-S and used it for real-time monitoring of HOCl production in neutrophil phagosomes. We show that it also reacts rapidly with hypobromous acid, bromamines, and hypoiodous acid, indicating that R19-S responds to these reactive halogen species as well as HOCl. Hypothiocyanous acid and taurine chloramine were unreactive, however, and ammonia chloramine and dichloramine reacted only very slowly. MS analyses revealed additional products from the reaction of HOCl with R19-S, including a chlorinated species as a minor product. Of note, phagocytosis of opsonized zymosan or Staphylococcus aureus by neutrophils was accompanied by an increase in R19 fluorescence. This increase depended on NADPH oxidase and myeloperoxidase activities, and detection of chlorinated R19-S confirmed its specificity for HOCl. Using live-cell imaging to track individual phagosomes in single neutrophils, we observed considerable heterogeneity among the phagosomes in the time from ingestion of a zymosan particle to when fluorescence was first detected, ranging from 1 to >30 min. However, once initiated, the subsequent fluorescence increase was uniform, reaching a similar maximum in ∼10 min. Our results confirm the utility of R19-S for detecting HOCl in real-time and provide definitive evidence that isolated neutrophils produce HOCl in phagosomes. The intriguing variability in the onset of HOCl production among phagosomes identified here could influence the way they kill ingested bacteria.

Associations between complement pathways activity, mannose-binding lectin, and odds of unprovoked venous thromboembolism.

INTRODUCTION: Deep vein thrombosis (DVT) originates in the valvular sinuses of large veins in a local milieu characterized by stasis and severe hypoxia. This may induce complement- and coagulation activation, which potentially increases the risk of venous thromboembolism (VTE). The aim of the present study was to investigate whether the activity of the complement pathways, the level of mannose-binding lectin (MBL) and tissue-factor (TF) induced thrombin generation were associated with risk of unprovoked VTE. METHODS: A case-control study was performed in patients with unprovoked VTE (n = 24) and age- and sex-matched healthy controls (n = 24). Serum complement pathway activity was measured by the total complement screen assay (Wieslab®). MBL was quantified by ELISA. Plasma TF-induced thrombin generation was measured using the CAT-assay. RESULTS: Activity in the highest quintile of the classical pathway was associated with increased odds of unprovoked VTE (OR 4.5, 95% CI; 0.8-24.7). Moreover, MBL deficiency (≤100 ng/ml) was associated with unprovoked VTE (OR 3.5, 95% Cl; 0.8-15.3). VTE patients had shortened TF-induced lag-time (4.8 ±â€¯0.6 min vs. 5.8 ±â€¯2.1 min, p < 0.001) and a higher endogenous thrombin potential (ETP) (1383 ±â€¯267 nM∗h vs. 1265 ±â€¯247 nM∗h, p = 0.07) than controls. No association between the classical complement pathway activity or MBL deficiency, and parameters of TF-induced thrombin generation was observed. CONCLUSION: Our findings suggest that high activity of the classical complement pathway, and MBL deficiency, might be associated with an increased odds of unprovoked VTE, independent of activation of TF-induced coagulation.

Involvement of pentraxin-3 in anti-neutrophil cytoplasmic antibody production induced by aluminum salt adjuvant.

OBJECTIVES: Pentraxin 3 (PTX3) is a multifunctional soluble factor. PTX3 can be involved in the regulation of vasculitis and is expressed in the cytoplasm of neutrophils. As anti-neutrophil cytoplasmic antibody (ANCA) is recognised as a cause of vasculitis, we aimed to discover the role of PTX3 in ANCA production in vivo. METHODS: To this end, we used aluminum salt (alum), which induces neutrophil extracellular traps, as an adjuvant for producing anti-myeloperoxidase-ANCA (MPO-ANCA). Specifically, we intraperitoneally injected alum and recombinant MPO (rMPO) into MPO-deficient mice and then measured the concentration of anti-MPO IgG in their blood. To show the involvement of extracellular PTX3 in this model, we assessed PTX3 protein content and host double-stranded DNA levels in the mice's peritoneal fluid after alum injection. In addition, we simultaneously administered recombinant PTX3, rMPO and alum to MPO-deficient mice to assess the function of PTX3 in producing anti-MPO IgG in vivo. RESULTS: Anti-MPO IgG was produced by the alum + rMPO immunisation model in MPO-deficient but not wildtype mice. Injection of alum induced extracellular PTX3 as well as double-stranded DNA and dead cells in MPO-deficient mice. Simultaneous injection of recombinant PTX3 with rMPO and alum attenuated the production of anti-MPO IgG in MPO-deficient mice. CONCLUSIONS: Our current findings provide evidence that PTX3 attenuates the production of murine MPO-ANCA.

The liver is a metabolic and immunologic organ: A reconsideration of metabolic decompensation due to infection in inborn errors of metabolism (IEM).

Metabolic decompensation in inborn errors of metabolism (IEM) is characterized by a rapid deterioration in metabolic status leading to life-threatening biochemical perturbations (e.g. hypoglycemia, hyperammonemia, acidosis, organ failure). Infection is the major cause of metabolic decompensation in patients with IEM. We hypothesized that activation of the immune system during infection leads to further perturbations in end-organ metabolism resulting in increased morbidity. To address this, we established model systems of metabolic decompensation due to infection. Using these systems, we have described the pathologic mechanisms of metabolic decompensation as well as changes in hepatic metabolic reserve associated with infection. First and foremost, our studies have demonstrated that the liver experiences a significant local innate immune response during influenza infection that modulates hepatic metabolism. Based on these findings, we are the first to suggest that the role of the liver as a metabolic and immunologic organ is central in the pathophysiology of metabolic decompensation due to infection in IEM. The dual function of the liver as a major metabolic regulator and a lymphoid organ responsible for immunosurveillance places this organ at risk for hepatotoxicity. Mobilization of hepatic reserve and the regenerative capacity of a healthy liver compensates for this calculated risk. However, activation of the hepatic innate immune system may be deleterious in IEM. Based on this assertion, strategies aimed at modulating the innate immune response may be a viable target for intervention in the treatment of hepatic metabolic decompensation.

Recurrent respiratory tract infections (RRTI) in the elderly: A late onset mild immunodeficiency?

Elderly with late-onset recurrent respiratory tract infections (RRTI) often have specific anti-polysaccharide antibody deficiency (SPAD). We hypothesized that late-onset RRTI is caused by mild immunodeficiencies, such as SPAD, that remain hidden through adult life. We analyzed seventeen elderly RRTI patients and matched controls. We determined lymphocyte subsets, expression of BAFF receptors, serum immunoglobulins, complement pathways, Pneumovax-23 vaccination response and genetic variations in BAFFR and MBL2. Twelve patients (71%) and ten controls (59%) had SPAD. IgA was lower in patients than in controls, but other parameters did not differ. However, a high percentage of both patients (53%) and controls (65%) were MBL deficient, much more than in the general population. Often, MBL2 secretor genotypes did not match functional deficiency, suggesting that functional MBL deficiency can be an acquired condition. In conclusion, we found SPAD and MBL deficiency in many elderly, and conjecture that at least the latter arises with age.

Insight on the impacts of free amino acids and their metabolites on the immune system from a perspective of inborn errors of amino acid metabolism.

INTRODUCTION: Amino acids (AAs) support a broad range of functions in living organisms, including several that affect the immune system. The functions of the immune system are affected when free AAs are depleted or in excess because of external factors, such as starvation, or because of genetic factors, such as inborn errors of metabolism. Areas covered: In this review, we discuss the current insights into how free AAs affect immune responses. When possible, we make comparisons to known disease states resulting from inborn errors of metabolism, in which changed levels of AAs or AA metabolites provide insight into the impact of AAs on the human immune system in vivo. We also explore the literature describing how changes in AA levels might provide pharmaceutical targets for safe immunomodulatory treatment. Expert opinion: The impact of free AAs on the immune system is a neglected topic in most immunology textbooks. That neglect is undeserved, because free AAs have both direct and indirect effects on the immune system. Consistent choices of pre-clinical models and better strategies for creating formulations are required to gain clinical impact.

Association between TLR2/TLR4 gene polymorphisms and COPD phenotype in a Greek cohort.

BACKGROUND: Considering that the innate immune system plays a pivotal role in the pathogenesis of chronic obstructive pulmonary disease (COPD), we hypothesized that functional single-nucleotide polymorphisms (SNPs) of innate immune genes affect the disease phenotype and prognosis. AIM: To elucidate the contribution of common functional TLR2 and TLR4 SNPs and genotypic deficiency of the mannose-binding lectin (MBL) protein, both as single parameters and in combination, in Greek COPD patients. RESULTS: In a cohort of 114 Greek COPD patients, we confirmed that the presence of TLR4-D299G or TLR4-T399I SNPs was significantly associated with an earlier COPD stage (p = 0.003 and p = 0.009, respectively). In comparison, the absence of any analyzed polymorphism, including those of TLR2-R753Q and genotypic MBL deficiency, was significantly associated with a more severe disease phenotype, characterized by more frequent exacerbations (p = 0.045). CONCLUSION: Our findings support the notion that the presence of innate immune SNPs, such as functional polymorphisms of TLRs along with MBL deficiency, might exert a protective effect on the COPD phenotype, similar with other immune-mediated disorders.

Cytokine-Induced Glucocorticoid Resistance from Eosinophil Activation: Protein Phosphatase 5 Modulation of Glucocorticoid Receptor Phosphorylation and Signaling.

The mechanisms contributing to persistent eosinophil activation and poor eosinopenic response to glucocorticoids in severe asthma are poorly defined. We examined the effect of cytokines typically overexpressed in the asthmatic airways on glucocorticoid signaling in in vitro activated eosinophils. An annexin V assay used to measure eosinophil apoptosis showed that cytokine combinations of IL-2 plus IL-4 as well as TNF-α plus IFN-γ, or IL-3, GM-CSF, and IL-5 alone significantly diminished the proapoptotic response to dexamethasone. We found that IL-2 plus IL-4 resulted in impaired phosphorylation and function of the nuclear glucocorticoid receptor (GCR). Proteomic analysis of steroid sensitive and resistant eosinophils identified several differentially expressed proteins, namely protein phosphatase 5 (PP5), formyl peptide receptor 2, and annexin 1. Furthermore, increased phosphatase activity of PP5 correlated with impaired phosphorylation of the GCR. Importantly, suppression of PP5 expression with small interfering RNA restored proper phosphorylation and the proapoptotic function of the GCR. We also examined the effect of lipoxin A4 on PP5 activation by IL-2 plus IL-4. Similar to PP5 small interfering RNA inhibition, pretreatment of eosinophils with lipoxin A4 restored GCR phosphorylation and the proaptoptotic function of GCs. Taken together, our results showed 1) a critical role for PP5 in cytokine-induced resistance to GC-mediated eosinophil death, 2) supported the dependence of GCR phosphorylation on PP5 activity, and 3) revealed that PP5 is a target of the lipoxin A4-induced pathway countering cytokine-induced resistance to GCs in eosinophils.