Publications by authors named "Mihai G Netea"

855 Publications

Understanding the increased risk of infections in diabetes: innate and adaptive immune responses in type 1 diabetes.

Metabolism 2021 May 7:154795. Epub 2021 May 7.

Department of Internal Medicine (463), Radboud University Medical Center, PO Box 9101, 6500, HB, Nijmegen, the Netherlands.

Aims: Patients with diabetes have a higher incidence of infections with Candida albicans, Staphylococcus aureus and Mycobacterium tuberculosis, yet factors contributing to this increased risk are largely unknown. We hypothesize that altered innate and adaptive immune responses during diabetes contribute to an increased susceptibility to infections.

Materials And Methods: We studied cytokine responses to ex vivo pathogenic stimulations in a cohort with type 1 diabetes (n = 243) and non-diabetic healthy control subjects (n = 56) using isolated peripheral blood mononuclear cells (PBMCs). Clinical phenotypical data including BMI, duration of diabetes, and HbA levels were collected and related to the cytokine production capacity.

Results: Adjusted for age, sex and BMI, the presence of diabetes was associated with significantly lower IL-1β, IL-6, TNF-α, and IL-17 production upon ex vivo stimulation of PBMCs with C. albicans and S. aureus (all, p < 0.05). In response to stimulation with M. tuberculosis only IL-17 (p < 0.001) was lower in patients with diabetes. Patients with the shortest diabetes duration had a significant lower IL-1β, IL-6 and TNF-α production (all, p < 0.01) after M. tuberculosis stimulation. Older patients had a significant lower IFN-γ (p < 0.05) production after stimulation with all three pathogens. HbA levels and BMI had no significant impact on cytokine production.

Conclusions: PBMCs of patients with type 1 diabetes demonstrate significantly lower cytokine production in response to stimulation with several pathogens, which likely explain, at least in part, the increased susceptibility for these infections.
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http://dx.doi.org/10.1016/j.metabol.2021.154795DOI Listing
May 2021

The COVID-19 puzzle: deciphering pathophysiology and phenotypes of a new disease entity.

Lancet Respir Med 2021 May 6. Epub 2021 May 6.

Department of Medical and Surgical Sciences for Children and Adults, University of Modena and Reggio Emilia, Modena, Italy.

The zoonotic SARS-CoV-2 virus that causes COVID-19 continues to spread worldwide, with devastating consequences. While the medical community has gained insight into the epidemiology of COVID-19, important questions remain about the clinical complexities and underlying mechanisms of disease phenotypes. Severe COVID-19 most commonly involves respiratory manifestations, although other systems are also affected, and acute disease is often followed by protracted complications. Such complex manifestations suggest that SARS-CoV-2 dysregulates the host response, triggering wide-ranging immuno-inflammatory, thrombotic, and parenchymal derangements. We review the intricacies of COVID-19 pathophysiology, its various phenotypes, and the anti-SARS-CoV-2 host response at the humoral and cellular levels. Some similarities exist between COVID-19 and respiratory failure of other origins, but evidence for many distinctive mechanistic features indicates that COVID-19 constitutes a new disease entity, with emerging data suggesting involvement of an endotheliopathy-centred pathophysiology. Further research, combining basic and clinical studies, is needed to advance understanding of pathophysiological mechanisms and to characterise immuno-inflammatory derangements across the range of phenotypes to enable optimum care for patients with COVID-19.
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http://dx.doi.org/10.1016/S2213-2600(21)00218-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8102044PMC
May 2021

The impact of the Fungus-Host-Microbiota interplay upon Candida albicans infections: current knowledge and new perspectives.

FEMS Microbiol Rev 2021 May;45(3)

NEXBIOME Therapeutics, 22 allée Alan Turing, 63000 Clermont-Ferrand, France.

Candida albicans is a major fungal pathogen of humans. It exists as a commensal in the oral cavity, gut or genital tract of most individuals, constrained by the local microbiota, epithelial barriers and immune defences. Their perturbation can lead to fungal outgrowth and the development of mucosal infections such as oropharyngeal or vulvovaginal candidiasis, and patients with compromised immunity are susceptible to life-threatening systemic infections. The importance of the interplay between fungus, host and microbiota in driving the transition from C. albicans commensalism to pathogenicity is widely appreciated. However, the complexity of these interactions, and the significant impact of fungal, host and microbiota variability upon disease severity and outcome, are less well understood. Therefore, we summarise the features of the fungus that promote infection, and how genetic variation between clinical isolates influences pathogenicity. We discuss antifungal immunity, how this differs between mucosae, and how individual variation influences a person's susceptibility to infection. Also, we describe factors that influence the composition of gut, oral and vaginal microbiotas, and how these affect fungal colonisation and antifungal immunity. We argue that a detailed understanding of these variables, which underlie fungal-host-microbiota interactions, will present opportunities for directed antifungal therapies that benefit vulnerable patients.
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http://dx.doi.org/10.1093/femsre/fuaa060DOI Listing
May 2021

The Architecture of Circulating Immune Cells Is Dysregulated in People Living With HIV on Long Term Antiretroviral Treatment and Relates With Markers of the HIV-1 Reservoir, Cytomegalovirus, and Microbial Translocation.

Front Immunol 2021 19;12:661990. Epub 2021 Apr 19.

Department of Internal Medicine and Radboud Center for Infectious Diseases, Radboud University Medical Center, Nijmegen, Netherlands.

Long-term changes in the immune system of successfully treated people living with HIV (PLHIV) remain incompletely understood. In this study, we assessed 108 white blood cell (WBC) populations in a cohort of 211 PLHIV on stable antiretroviral therapy and in 56 HIV-uninfected controls using flow cytometry. We show that marked differences exist in T cell maturation and differentiation between PLHIV and HIV-uninfected controls: PLHIV had reduced percentages of CD4+ T cells and naïve T cells and increased percentages of CD8+ T cells, effector T cells, and T helper 17 (Th17) cells, together with increased Th17/regulatory T cell (Treg) ratios. PLHIV also exhibited altered B cell maturation with reduced percentages of memory B cells and increased numbers of plasmablasts. Determinants of the T and B cell composition in PLHIV included host factors (age, sex, and smoking), markers of the HIV reservoir, and CMV serostatus. Moreover, higher circulating Th17 percentages were associated with higher plasma concentrations of interleukin (IL) 6, soluble CD14, the gut homing chemokine CCL20, and intestinal fatty acid binding protein (IFABP). The changes in circulating lymphocytes translated into functional changes with reduced interferon (IFN)- γ responses of peripheral blood mononuclear cells to stimulation with and  In conclusion, this comprehensive analysis confirms the importance of persistent abnormalities in the number and function of circulating immune cells in PLHIV on stable treatment.
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http://dx.doi.org/10.3389/fimmu.2021.661990DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8091964PMC
April 2021

Glutathione Metabolism Contributes to the Induction of Trained Immunity.

Cells 2021 Apr 21;10(5). Epub 2021 Apr 21.

Department of Internal Medicine and Radboud Center for Infectious Diseases (RCI), Radboud University Nijmegen Medical Center, 6500 HB Nijmegen, The Netherlands.

The innate immune system displays heterologous memory characteristics, which are characterized by stronger responses to a secondary challenge. This phenomenon termed trained immunity relies on epigenetic and metabolic rewiring of innate immune cells. As reactive oxygen species (ROS) production has been associated with the trained immunity phenotype, we hypothesized that the increased ROS levels and the main intracellular redox molecule glutathione play a role in the induction of trained immunity. Here we show that pharmacological inhibition of ROS in an in vitro model of trained immunity did not influence cell responsiveness; the modulation of glutathione levels reduced pro-inflammatory cytokine production in human monocytes. Single nucleotide polymorphisms (SNPs) in genes involved in glutathione metabolism were found to be associated with changes in pro-inflammatory cytokine production capacity upon trained immunity. Also, plasma glutathione concentrations were positively associated with ex vivo IL-1β production, a biomarker of trained immunity, produced by monocytes of BCG-vaccinated individuals. In conclusion, glutathione metabolism is involved in the induction of trained immunity, and future studies are warranted to explore its functional consequences in human diseases.
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http://dx.doi.org/10.3390/cells10050971DOI Listing
April 2021

Conceptualization of population-specific human functional immune-genomics projects to identify factors that contribute to variability in immune and infectious diseases.

Heliyon 2021 Apr 13;7(4):e06755. Epub 2021 Apr 13.

Department of Internal Medicine and Radboud Center for Infectious Diseases (RCI), Radboud University Medical Center, Nijmegen, 6525 HP, the Netherlands.

The human immune system presents remarkable inter-individual variability in response to pathogens or perturbations. Recent high-throughput technologies have enabled the identification of both heritable and non-heritable determinants of immune response variation between individuals. In this review, we summarize the advances made through the Human Functional Genomics Projects (HFGPs), challenges and the need for more refined strategies. Inter-individual variability in stimulation-induced cytokine responses is influenced in part by age, gender, seasonality, and gut microbiome. Host genetic regulators especially single nucleotide polymorphisms in multiple immune gene loci, particularly the TLR1-TLR6-TLR10 locus, have been identified using individuals of predominantly European descent. However, transferability of such findings to other populations is challenging. We are beginning to incorporate diverse population cohorts and leverage multi-omics approaches at single cell level to bridge the current knowledge gap. We believe that such an approach presents the opportunities to comprehensively assess both genetic and environmental factors driving variation seen in immune response phenotype and a better understanding of the molecular and biological mechanisms involved.
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http://dx.doi.org/10.1016/j.heliyon.2021.e06755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8066384PMC
April 2021

Immune cells fold and damage fungal hyphae.

Proc Natl Acad Sci U S A 2021 Apr;118(15)

Aberdeen Fungal Group, Institute of Medical Sciences, Foresterhill, AB25 2ZD Aberdeen, United Kingdom;

Innate immunity provides essential protection against life-threatening fungal infections. However, the outcomes of individual skirmishes between immune cells and fungal pathogens are not a foregone conclusion because some pathogens have evolved mechanisms to evade phagocytic recognition, engulfment, and killing. For example, can escape phagocytosis by activating cellular morphogenesis to form lengthy hyphae that are challenging to engulf. Through live imaging of -macrophage interactions, we discovered that macrophages can counteract this by folding fungal hyphae. The folding of fungal hyphae is promoted by Dectin-1, β2-integrin, VASP, actin-myosin polymerization, and cell motility. Folding facilitates the complete engulfment of long hyphae in some cases and it inhibits hyphal growth, presumably tipping the balance toward successful fungal clearance.
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http://dx.doi.org/10.1073/pnas.2020484118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8053999PMC
April 2021

Data of common and species-specific transcriptional host responses to pathogenic fungi.

Data Brief 2021 Apr 4;35:106928. Epub 2021 Mar 4.

Department of Internal Medicine and Radboudumc Center for Infectious Diseases (RCI), Radboud University Medical Center, Nijmegen, The Netherlands.

Using a comparative RNA-Sequencing based transcriptional profiling approach, responses of primary human peripheral blood mononuclear cells (PBMCs) to common human pathogenic fungi have been characterized (Bruno et al. Computational and Structural Biology Journal). Primary human PBMCs were stimulated in vitro with the fungi , and after which RNA was isolated and sequenced. From raw sequencing reads differential expressed genes in response to the different fungi where calculated by comparison with unstimulated cells. By overlapping differentially expressed genes in response to the pathogenic fungi , and a dataset was generated that encompasses a common response to these three distinct fungi as well as species-specific responses. Here we present datasets on these common and species-specific responses that complement the original study (Bruno et al. Computational and Structural Biology Journal). These data serve to facilitate further fundamental research on the immune response to opportunistic pathogenic fungi such as , and .
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http://dx.doi.org/10.1016/j.dib.2021.106928DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8039545PMC
April 2021

The anti-inflammatory cytokine interleukin-37 is an inhibitor of trained immunity.

Cell Rep 2021 Apr;35(1):108955

Department of Medicine, Radboud University Medical Center, Nijmegen, the Netherlands; Department of Medicine, University of Colorado Denver, Aurora, CO 80045, USA. Electronic address:

Trained immunity (TI) is a de facto innate immune memory program induced in monocytes/macrophages by exposure to pathogens or vaccines, which evolved as protection against infections. TI is characterized by immunometabolic changes and histone post-translational modifications, which enhance production of pro-inflammatory cytokines. As aberrant activation of TI is implicated in inflammatory diseases, tight regulation is critical; however, the mechanisms responsible for this modulation remain elusive. Interleukin-37 (IL-37) is an anti-inflammatory cytokine that curbs inflammation and modulates metabolic pathways. In this study, we show that administration of recombinant IL-37 abrogates the protective effects of TI in vivo, as revealed by reduced host pro-inflammatory responses and survival to disseminated candidiasis. Mechanistically, IL-37 reverses the immunometabolic changes and histone post-translational modifications characteristic of TI in monocytes, thus suppressing cytokine production in response to infection. IL-37 thereby emerges as an inhibitor of TI and as a potential therapeutic target in immune-mediated pathologies.
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http://dx.doi.org/10.1016/j.celrep.2021.108955DOI Listing
April 2021

Bridging animal and clinical research during SARS-CoV-2 pandemic: A new-old challenge.

EBioMedicine 2021 Apr 1;66:103291. Epub 2021 Apr 1.

Ludwig Boltzmann Institute for Experimental and Clinical Traumatology in the AUVA Research Center, Donaueschingenstrasse 13, 1200, Vienna, Austria. Electronic address:

Many milestones in medical history rest on animal modeling of human diseases. The SARS-CoV-2 pandemic has evoked a tremendous investigative effort primarily centered on clinical studies. However, several animal SARS-CoV-2/COVID-19 models have been developed and pre-clinical findings aimed at supporting clinical evidence rapidly emerge. In this review, we characterize the existing animal models exposing their relevance and limitations as well as outline their utility in COVID-19 drug and vaccine development. Concurrently, we summarize the status of clinical trial research and discuss the novel tactics utilized in the largest multi-center trials aiming to accelerate generation of reliable results that may subsequently shape COVID-19 clinical treatment practices. We also highlight areas of improvement for animal studies in order to elevate their translational utility. In pandemics, to optimize the use of strained resources in a short time-frame, optimizing and strengthening the synergy between the preclinical and clinical domains is pivotal.
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http://dx.doi.org/10.1016/j.ebiom.2021.103291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8016444PMC
April 2021

Polymorphisms within Autophagy-Related Genes Influence the Risk of Developing Colorectal Cancer: A Meta-Analysis of Four Large Cohorts.

Cancers (Basel) 2021 Mar 12;13(6). Epub 2021 Mar 12.

Division of Molecular Genetic Epidemiology, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.

The role of genetic variation in autophagy-related genes in modulating autophagy and cancer is poorly understood. Here, we comprehensively investigated the association of autophagy-related variants with colorectal cancer (CRC) risk and provide new insights about the molecular mechanisms underlying the associations. After meta-analysis of the genome-wide association study (GWAS) data from four independent European cohorts (8006 CRC cases and 7070 controls), two loci, ( = 2.19 × 10) and ( = 6.28 × 10) were associated with the risk of CRC. Mechanistically, the allele was associated with IL1 β levels after the stimulation of peripheral blood mononuclear cells (PBMCs) with ( = 0.002), CD24 + CD38 + CD27 + IgM + B cell levels in blood ( = 0.0038) and serum levels of en-RAGE ( = 0.0068). allele was associated with TNF α and IL1 β levels after the stimulation of PBMCs with LPS ( = 0.0088 and = 0.0076, respectively), CD14+CD16- cell levels in blood ( = 0.0068) and serum levels of CCL19 and cortisol ( = 0.0052 and = 0.0074, respectively). Interestingly, no association with autophagy flux was observed. These results suggested an effect of the and loci in the pathogenesis of CRC, likely through the modulation of host immune responses.
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http://dx.doi.org/10.3390/cancers13061258DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7998818PMC
March 2021

Analysis of HLA gene polymorphisms in East Africans reveals evidence of gene flow in two Semitic populations from Sudan.

Eur J Hum Genet 2021 Mar 22. Epub 2021 Mar 22.

Human Genetics Laboratory, Department of Genomics and Evolutionary Biology, National Institute of Genetics, Mishima, Shizuoka, Japan.

Sudan, a northeastern African country, is characterized by high levels of cultural, linguistic, and genetic diversity, which is believed to be affected by continuous migration from neighboring countries. Consistent with such demographic effect, genome-wide SNP data revealed a shared ancestral component among Sudanese Afro-Asiatic speaking groups and non-African populations, mainly from West Asia. Although this component is shared among all Afro-Asiatic speaking groups, the extent of this sharing in Semitic groups, such as Sudanese Arab, is still unknown. Using genotypes of six polymorphic human leukocyte antigen (HLA) genes (i.e., HLA-A, -C, -B, -DRB1, -DQB1, and -DPB1), we examined the genetic structure of eight East African ethnic groups with origins in Sudan, South Sudan, and Ethiopia. We identified informative HLA alleles using principal component analysis, which revealed that the two Semitic groups (Gaalien and Shokrya) constituted a distinct cluster from the other Afro-Asiatic speaking groups in this study. The HLA alleles that distinguished Semitic Arabs co-exist in the same extended HLA haplotype, and those alleles are in strong linkage disequilibrium. Interestingly, we find the four-locus haplotype "C*12:02-B*52:01-DRB1*15:02-DQB1*06:01" exclusively in non-African populations and it is widely spread across Asia. The identification of this haplotype suggests a gene flow from Asia, and likely these haplotypes were brought to Africa through back migration from the Near East. These findings will be of interest to biomedical and anthropological studies that examine the demographic history of northeast Africa.
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http://dx.doi.org/10.1038/s41431-021-00845-6DOI Listing
March 2021

Rare variants increase the risk of severe COVID-19.

Elife 2021 Mar 23;10. Epub 2021 Mar 23.

Department of Internal Medicine, Radboudumc Center for Infectious Diseases, Nijmegen, Netherlands.

Evidence is mounting that rare loss-of-function variants in the gene predispose men with no medical history to severe forms of COVID-19.
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http://dx.doi.org/10.7554/eLife.67860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7987329PMC
March 2021

Immunomodulatory aged neutrophils are augmented in blood and skin of psoriasis patients.

J Allergy Clin Immunol 2021 Mar 19. Epub 2021 Mar 19.

Department of Laboratory Medicine, Laboratory of Medical Immunology, Radboudumc, Nijmegen, The Netherlands. Electronic address:

Background: Neutrophil accumulation in the skin is a hallmark of psoriasis. Novel insights on neutrophil phenotypic and functional heterogeneity raise the question to what extent these cells contribute to the sustained inflammatory skin reaction.

Objective: We sought to examine the phenotype and functional properties of neutrophils in blood and skin of patients with psoriasis, and the effect of TNF-α and p40(IL-12/IL-23) antibody therapy on circulating neutrophils.

Methods: Thirty-two patients with psoriasis were enrolled in an observational study performed in 2 university hospitals. We evaluated neutrophil phenotype and function using in vitro (co)culture stimulation assays, flow cytometry, multiplex immunohistochemistry, and multispectral imaging of patient-derived blood and skin samples.

Results: Cluster of differentiation (CD)10 and CD10 neutrophils were increased in peripheral blood of patients with psoriasis. In CD10 neutrophils, different maturation stages were observed, including a subset resembling aged neutrophils that was 3 times more abundant than in healthy individuals. These aged neutrophils displayed suboptimal canonical neutrophil functions and induced IL-17 and IFN-γ production by T cells in vitro, mediated by neutrophil extracellular trap formation. Also, mature and aged neutrophils were present in psoriatic skin and were found in the vicinity of T cells. Upon antibody therapy, numbers of these cells in circulation decreased.

Conclusions: Patients with psoriasis reveal a unique neutrophil profile in circulation, and 2 distinct neutrophil subsets are present in psoriatic skin. Targeted biological treatment may aid in the containment of sustained neutrophil-mediated inflammation.
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http://dx.doi.org/10.1016/j.jaci.2021.02.041DOI Listing
March 2021

Long-term treated HIV infection is associated with platelet mitochondrial dysfunction.

Sci Rep 2021 Mar 18;11(1):6246. Epub 2021 Mar 18.

Department of Internal Medicine, Radboud Center for Infectious Diseases, Radboud University Medical Center, 6500 HB, Nijmegen, The Netherlands.

HIV infection and antiretroviral therapy have been linked to mitochondrial dysfunction. The role of platelet mitochondrial dysfunction in thrombosis, immunoregulation and age-related diseases is increasingly appreciated. Here, we studied platelet mitochondrial DNA content (mtDNA) and mitochondrial function in people living with HIV (PLHIV) and related this to platelet function. In a cohort of 208 treated PLHIV and 56 uninfected controls, mtDNA was quantified, as well as platelet activation, platelet agonist-induced reactivity and inflammation by circulating factors and flow cytometry. In a subgroup of participants, the metabolic activity of platelets was further studied by mitochondrial function tests and the Seahorse Flux Analyzer. PLHIV had significantly lower mtDNA compared to controls (8.5 copies/platelet (IQR: 7.0-10.7) vs. 12.2 copies/platelet (IQR: 9.5-16.6); p < 0.001), also after correction for age, sex and BMI. Prior zidovudine-use (n = 46) was associated with a trend for lower mtDNA. PLHIV also had reduced ex vivo platelet reactivity and mean platelet volume compared to controls. MtDNA correlated positively with both platelet parameters and correlated negatively with inflammatory marker sCD163. Mitochondrial function tests in a subgroup of participants confirmed the presence of platelet mitochondrial respiration defects. Platelet mitochondrial function is disturbed in PLHIV, which may contribute to platelet dysfunction and subsequent complications. Interventions targeting the preservation of normal platelet mitochondrial function may ultimately prove beneficial for PLHIV.
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http://dx.doi.org/10.1038/s41598-021-85775-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7973809PMC
March 2021

induction of trained immunity in adherent human monocytes.

STAR Protoc 2021 Mar 24;2(1):100365. Epub 2021 Feb 24.

Department of Internal Medicine, Radboud University Nijmegen Medical Centre, Geert Grooteplein 8, 6500HB Nijmegen, the Netherlands.

A growing number of studies show that innate immune cells can undergo functional reprogramming, facilitating a faster and enhanced response to heterologous secondary stimuli. This concept has been termed "trained immunity." We outline here a protocol to recapitulate this using adherent monocytes from consecutive isolation of peripheral blood mononuclear cells. The induction of trained immunity and the associated functional reprogramming of monocytes is described in detail using β-glucan (from ) and Bacillus Calmette-Guérin as examples. For complete details on the use and execution of this protocol, please refer to Repnik et al. (2003) and Bekkering et al. (2016).
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http://dx.doi.org/10.1016/j.xpro.2021.100365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7921712PMC
March 2021

The association of TSH and thyroid hormones with lymphopenia in bacterial sepsis and COVID-19.

J Clin Endocrinol Metab 2021 Mar 13. Epub 2021 Mar 13.

Department of Internal Medicine, Division of Endocrinology, Radboud University Nijmegen, GA, Nijmegen, the Netherlands.

Context: Lymphopenia is a key feature of immune dysfunction in patients with bacterial sepsis and COVID-19 and associated with poor clinical outcomes, but the cause is largely unknown. Severely ill patients may present with thyroid function abnormalities, so-called non-thyroidal illness syndrome (NTIS), and several studies have linked TSH and the thyroid hormones thyroxine (T4) and triiodothyronine (T3) to homeostatic regulation and function of lymphocyte populations.

Purpose: To test the hypothesis that abnormal thyroid function correlates with lymphopenia in patients with severe infections.

Methods: Retrospective analysis of absolute lymphocyte counts, circulating TSH, T4, free T4 (FT4), T3, albumin and inflammatory biomarkers was performed in two independent hospitalized study populations: bacterial sepsis (n=224) and COVID-19 patients (n=161). A subgroup analysis was performed in patients with severe lymphopenia and normal lymphocyte counts.

Results: Only T3 significantly correlated (rho=0.252) with lymphocyte counts in patients with bacterial sepsis and lower concentrations were found in severe lymphopenic compared to non-lympopenic patients (n=56 per group). Severe lymphopenic COVID-19 patients (n=17) showed significantly lower plasma concentrations of TSH, T4, FT4 and T3 compared to patients without lymphopenia (n=18), and demonstrated significantly increased values of the inflammatory markers interleukin-6, C-reactive protein and ferritin. Remarkably, after one week follow-up, the majority (12/15) of COVID-19 patients showed quantitative recovery of their lymphocyte numbers, while TSH and thyroid hormones remained mainly disturbed.

Conclusion: Abnormal thyroid function correlates with lymphopenia in patients with severe infections, like bacterial sepsis and COVID-19, but future studies need to establish whether a causal relationship is involved.
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http://dx.doi.org/10.1210/clinem/dgab148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7989224PMC
March 2021

Lysine methyltransferase G9a is an important modulator of trained immunity.

Clin Transl Immunology 2021 18;10(2):e1253. Epub 2021 Feb 18.

Department of Internal Medicine Radboud Center for Infectious Diseases (RCI) Radboud University Medical Center Nijmegen The Netherlands.

Objectives: Histone methyltransferase G9a, also known as Euchromatic Histone Lysine Methyltransferase 2 (EHMT2), mediates H3K9 methylation which is associated with transcriptional repression. It possesses immunomodulatory effects and is overexpressed in multiple types of cancer. In this study, we investigated the role of G9a in the induction of trained immunity, a innate immune memory, and its effects in non-muscle-invasive bladder cancer (NMIBC) patients treated with intravesical Bacillus Calmette-Guérin (BCG).

Methods: EHMT2 expression was assessed upon induction of trained immunity by RNA sequencing and Western blotting. G9a inhibitor BIX-01294 was used to investigate the effect on trained immunity responses . Subsequent cytokine production was measured by ELISA, epigenetic modifications were measured by ChIP-qPCR, Seahorse technology was used to measure metabolic changes, and a luminescence assay was used to measure ROS release. RNA sequencing was performed on BIX-01294-treated monocytes .

Results: The expression of EHMT2 mRNA and protein decreased in monocytes during induction of trained immunity. G9a inhibition by BIX-01294 induced trained immunity and amplified trained immunity responses evoked by various microbial ligands . This was accompanied by decreased H3K9me2 at the promoters of pro-inflammatory genes. G9a inhibition was also associated with amplified trained immunity responses in circulating monocytes of NMIBC patients. Additionally, altered RNA expression of inflammatory genes in monocytes of NMIBC patients was observed upon G9a inhibition. Furthermore, intravesical BCG therapy decreased H3K9me2 at the promoter of pro-inflammatory genes.

Conclusion: Inhibition of G9a is important in the induction of trained immunity, and G9a may represent a novel therapeutic target in NMIBC patients.
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http://dx.doi.org/10.1002/cti2.1253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7890679PMC
February 2021

Prosaposin mediates inflammation in atherosclerosis.

Sci Transl Med 2021 Mar;13(584)

Department of Internal Medicine and Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, 6525 GA Nijmegen, Netherlands.

Macrophages play a central role in the pathogenesis of atherosclerosis. The inflammatory properties of these cells are dictated by their metabolism, of which the mechanistic target of rapamycin (mTOR) signaling pathway is a key regulator. Using myeloid cell-specific nanobiologics in apolipoprotein E-deficient () mice, we found that targeting the mTOR and ribosomal protein S6 kinase-1 (S6K1) signaling pathways rapidly diminished plaque macrophages' inflammatory activity. By investigating transcriptome modifications, we identified , a gene encoding the lysosomal protein prosaposin, as closely related with mTOR signaling. Subsequent in vitro experiments revealed that inhibition suppressed both glycolysis and oxidative phosphorylation. Transplantation of bone marrow to low-density lipoprotein receptor knockout () mice led to a reduction in atherosclerosis development and plaque inflammation. Last, we confirmed the relationship between expression and inflammation in human carotid atherosclerotic plaques. Our findings provide mechanistic insights into the development of atherosclerosis and identify prosaposin as a potential therapeutic target.
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http://dx.doi.org/10.1126/scitranslmed.abe1433DOI Listing
March 2021

An open label trial of anakinra to prevent respiratory failure in COVID-19.

Elife 2021 03 8;10. Epub 2021 Mar 8.

4th Department of Internal Medicine, National and Kapodistrian University of Athens, Athens, Greece.

Background: It was studied if early suPAR-guided anakinra treatment can prevent severe respiratory failure (SRF) of COVID-19.

Methods: A total of 130 patients with suPAR ≥6 ng/ml were assigned to subcutaneous anakinra 100 mg once daily for 10 days. Primary outcome was SRF incidence by day 14 defined as any respiratory ratio below 150 mmHg necessitating mechanical or non-invasive ventilation. Main secondary outcomes were 30-day mortality and inflammatory mediators; 28-day WHO-CPS was explored. Propensity-matched standard-of care comparators were studied.

Results: 22.3% with anakinra treatment and 59.2% comparators (hazard ratio, 0.30; 95% CI, 0.20-0.46) progressed into SRF; 30-day mortality was 11.5% and 22.3% respectively (hazard ratio 0.49; 95% CI 0.25-0.97). Anakinra was associated with decrease in circulating interleukin (IL)-6, sCD163 and sIL2-R; IL-10/IL-6 ratio on day 7 was inversely associated with SOFA score; patients were allocated to less severe WHO-CPS strata.

Conclusions: Early suPAR-guided anakinra decreased SRF and restored the pro-/anti-inflammatory balance.

Funding: This study was funded by the Hellenic Institute for the Study of Sepsis, Technomar Shipping Inc, Swedish Orphan Biovitrum, and the Horizon 2020 Framework Programme.

Clinical Trial Number: NCT04357366.
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http://dx.doi.org/10.7554/eLife.66125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8034977PMC
March 2021

A modular approach toward producing nanotherapeutics targeting the innate immune system.

Sci Adv 2021 Mar 5;7(10). Epub 2021 Mar 5.

Biomedical Engineering and Imaging Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

Immunotherapies controlling the adaptive immune system are firmly established, but regulating the innate immune system remains much less explored. The intrinsic interactions between nanoparticles and phagocytic myeloid cells make these materials especially suited for engaging the innate immune system. However, developing nanotherapeutics is an elaborate process. Here, we demonstrate a modular approach that facilitates efficiently incorporating a broad variety of drugs in a nanobiologic platform. Using a microfluidic formulation strategy, we produced apolipoprotein A1-based nanobiologics with favorable innate immune system-engaging properties as evaluated by in vivo screening. Subsequently, rapamycin and three small-molecule inhibitors were derivatized with lipophilic promoieties, ensuring their seamless incorporation and efficient retention in nanobiologics. A short regimen of intravenously administered rapamycin-loaded nanobiologics (mTORi-NBs) significantly prolonged allograft survival in a heart transplantation mouse model. Last, we studied mTORi-NB biodistribution in nonhuman primates by PET/MR imaging and evaluated its safety, paving the way for clinical translation.
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http://dx.doi.org/10.1126/sciadv.abe7853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7935355PMC
March 2021

Deadly COVID-19 among the elderly: Innate immune memory helping those most in need.

Med (N Y) 2021 Apr 24;2(4):378-383. Epub 2021 Feb 24.

Department of Internal Medicine and Radboud Centre of Infectious Diseases (RCI), Radboud University Medical Centre, Nijmegen, the Netherlands.

Age is a key risk factor associated with the severity of symptoms caused by SARS-CoV-2, and there is an urgent need to reduce COVID-19 morbidity and mortality in elderly individuals. We discuss evidence suggesting that trained immunity elicited by BCG vaccination may improve immune responses and can serve as a strategy to combat COVID-19 in this population.
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http://dx.doi.org/10.1016/j.medj.2021.02.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7903897PMC
April 2021

Trained Immunity: Reprogramming Innate Immunity in Health and Disease.

Annu Rev Immunol 2021 Apr 26;39:667-693. Epub 2021 Feb 26.

Department of Internal Medicine and Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, 6525 GA Nijmegen, Netherlands; email:

Traditionally, the innate and adaptive immune systems are differentiated by their specificity and memory capacity. In recent years, however, this paradigm has shifted: Cells of the innate immune system appear to be able to gain memory characteristics after transient stimulation, resulting in an enhanced response upon secondary challenge. This phenomenon has been called trained immunity. Trained immunity is characterized by nonspecific increased responsiveness, mediated via extensive metabolic and epigenetic reprogramming. Trained immunity explains the heterologous effects of vaccines, which result in increased protection against secondary infections. However, in chronic inflammatory conditions, trained immunity can induce maladaptive effects and contribute to hyperinflammation and progression of cardiovascular disease, autoinflammatory syndromes, and neuroinflammation. In this review we summarize the current state of the field of trained immunity, its mechanisms, and its roles in both health and disease.
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http://dx.doi.org/10.1146/annurev-immunol-102119-073855DOI Listing
April 2021

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

Chronic HIV infection induces transcriptional and functional reprogramming of innate immune cells.

JCI Insight 2021 Apr 8;6(7). Epub 2021 Apr 8.

Department of Internal Medicine, Radboud Center for Infectious Diseases, Radboud University Medical Center, Nijmegen, Netherlands.

Chronic inflammation and immune dysfunction play a key role in the development of non-AIDS-related comorbidities. The aim of our study was to characterize the functional phenotype of immune cells in people living with HIV (PLHIV). We enrolled a cross-sectional cohort study of PLHIV on stable antiretroviral therapy and healthy controls. We assessed ex vivo cytokine production capacity and transcriptomics of monocytes and T cells upon bacterial, fungal, and viral stimulation. PLHIV exhibited an exacerbated proinflammatory profile in monocyte-derived cytokines, but not in lymphocyte-derived cytokines. Particularly, the production of the IL-1β to imiquimod, E. coli LPS, and Mycobacterium tuberculosis was increased, and this production correlated with plasma concentrations of high-sensitivity C-reactive protein and soluble CD14. This increase in monocyte responsiveness remained stable over time in subsequent blood sampling after more than 1 year. Transcriptome analyses confirmed priming of the monocyte IL-1β pathway, consistent with a monocyte-trained immunity phenotype. Increased plasma concentrations of β-glucan, a well-known inducer of trained immunity, were associated with increased innate cytokine responses. Monocytes of PLHIV exhibited a sustained proinflammatory immune phenotype with priming of the IL-1β pathway. Training of the innate immune system in PLHIV likely plays a role in long-term HIV complications and provides a promising therapeutic target for inflammation-related comorbidities.
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http://dx.doi.org/10.1172/jci.insight.145928DOI Listing
April 2021

IL-38 prevents induction of trained immunity by inhibition of mTOR signaling.

J Leukoc Biol 2021 Feb 23. Epub 2021 Feb 23.

Department of Medicine, University of Colorado, Aurora, Colorado, USA.

Trained immunity is the acquisition of a hyperresponsive phenotype by innate immune cells (such as monocytes and macrophages) after an infection or vaccination, a de facto nonspecific memory dependent on epigenetic and metabolic reprogramming of these cells. We have recently shown that induction of trained immunity is dependent on IL-1β. Here, we show that recombinant IL-38, an anti-inflammatory cytokine of the IL-1-family, was able to induce long-term inhibitory changes and reduce the induction of trained immunity by β-glucan in vivo in C57BL/6 mice and ex vivo in their bone marrow cells. IL-38 blocked mTOR signaling and prevented the epigenetic and metabolic changes induced by β-glucan. In healthy subjects, the IL1F10 associated single nucleotide polymorphism rs58965312 correlated with higher plasma IL-38 concentrations and reduced induction of trained immunity by β-glucan ex vivo. These results indicate that IL-38 induces long-term anti-inflammatory changes and also inhibits the induction of trained immunity. Recombinant IL-38 could therefore potentially be used as a therapeutic intervention for diseases characterized by exacerbated trained immunity.
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http://dx.doi.org/10.1002/JLB.3A0220-143RRRDOI Listing
February 2021

Systemic administration of β-glucan induces immune training in microglia.

J Neuroinflammation 2021 Feb 22;18(1):57. Epub 2021 Feb 22.

Department of Biomedical Sciences of Cells & Systems, Section Molecular Neurobiology, University of Groningen, University Medical Center Groningen, Antonius Deusinglaan 1, 9713, AV, Groningen, The Netherlands.

Background: An innate immune memory response can manifest in two ways: immune training and immune tolerance, which refers to an enhanced or suppressed immune response to a second challenge, respectively. Exposing monocytes to moderate-to-high amounts of bacterial lipopolysaccharide (LPS) induces immune tolerance, whereas fungal β-glucan (BG) induces immune training. In microglia, it has been shown that different LPS inocula in vivo can induce either immune training or tolerance. Few studies focused on impact of BG on microglia and were only performed in vitro. The aim of the current study was to determine whether BG activates and induces immune memory in microglia upon peripheral administration in vivo.

Methods: Two experimental designs were used. In the acute design, mice received an intraperitoneal (i.p.) injection with PBS, 1 mg/kg LPS or 20 mg/kg BG and were terminated after 3 h, 1 or 2 days. In the preconditioning design, animals were first challenged i.p. with PBS, 1 mg/kg LPS or 20 mg/kg BG. After 2, 7 or 14 days, mice received a second injection with PBS or 1 mg/kg LPS and were sacrificed 3 h later. Microglia were isolated by fluorescence-activated cell sorting, and cytokine gene expression levels were determined. In addition, a self-developed program was used to analyze microglia morphological changes. Cytokine concentrations in serum were determined by a cytokine array.

Results: Microglia exhibited a classical inflammatory response to LPS, showing significant upregulation of Tnf, Il6, Il1β, Ccl2, Ccl3 and Csf1 expression, three h after injection, and obvious morphological changes 1 and 2 days after injection. With an interval of 2 days between two challenges, both BG and LPS induced immune training in microglia. The training effect of LPS changed into immune tolerance after a 7-day interval between 2 LPS challenges. Preconditioning with BG and LPS resulted in increased morphological changes in microglia in response to a systemic LPS challenge compared to naïve microglia.

Conclusions: Our results demonstrate that preconditioning with BG and LPS both induced immune training of microglia at two days after the first challenge. However, with an interval of 7 days between the first and second challenge, LPS-preconditioning resulted in immune tolerance in microglia.
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http://dx.doi.org/10.1186/s12974-021-02103-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901224PMC
February 2021

BCG-induced non-specific effects on heterologous infectious disease in Ugandan neonates: an investigator-blind randomised controlled trial.

Lancet Infect Dis 2021 Feb 17. Epub 2021 Feb 17.

Department of Infection Biology, London School of Hygiene and Tropical Medicine, London, UK; MRC/UVRI and LSHTM Uganda Research Unit, Entebbe, Uganda.

Background: Trials done in infants with low birthweight in west Africa suggest that BCG vaccination reduces all-cause mortality in the neonatal period, probably because of heterologous protection against non-tuberculous infections. This study investigated whether BCG alters all-cause infectious disease morbidity in healthy infants in a different high-mortality setting, and explored whether the changes are mediated via trained innate immunity.

Methods: This was an investigator-blind, randomised, controlled trial done at one hospital in Entebbe, Uganda. Infants who were born unwell (ie, those who were not well enough to be discharged directly home from the labour ward because they required medical intervention), with major congenital malformations, to mothers with HIV, into families with known or suspected tuberculosis, or for whom cord blood samples could not be taken, were excluded from the study. Any other infant well enough to be discharged directly from the labour ward was eligible for inclusion, with no limitation on gestational age or birthweight. Participants were recruited at birth and randomly assigned (1:1) to receive standard dose BCG 1331 (BCG-Danish) on the day of birth or at age 6 weeks (computer-generated randomisation, block sizes of 24, stratified by sex). Investigators and clinicians were masked to group assignment; parents were not masked. Participants were clinically followed up to age 10 weeks and contributed blood samples to one of three immunological substudies. The primary clinical outcome was physician-diagnosed non-tuberculous infectious disease incidence. Primary immunological outcomes were histone trimethylation at the promoter region of TNF, IL6, and IL1B; ex-vivo production of TNF, IL-6, IL-1β, IL-10, and IFNγ after heterologous stimulation; and transferrin saturation and hepcidin levels. All outcomes were analysed in the modified intention-to-treat population of all randomly assigned participants except those whose for whom consent was withdrawn. This trial is registered with the International Standard Randomised Controlled Trial Number registry (#59683017).

Findings: Between Sept 25, 2014, and July 31, 2015, 560 participants were enrolled and randomly assigned to receive BCG at birth (n=280) or age 6 weeks (n=280). 12 participants assigned to receive BCG at birth and 11 participants assigned to receive BCG at age 6 weeks were withdrawn from the study by their parents shortly after randomisation and were not included in analyses. During the first 6 weeks of life before the infants in the delayed vaccination group received BCG vaccination, physician-diagnosed non-tuberculous infectious disease incidence was lower in infants in the BCG at birth group than in the delayed group (98 presentations in the BCG at birth group vs 129 in the delayed BCG group; hazard ratio [HR] 0·71 [95% CI 0·53-0·95], p=0·023). After BCG in the delayed group (ie, during the age 6-10 weeks follow-up), there was no significant difference in non-tuberculous infectious disease incidence between the groups (88 presentations vs 76 presentations; HR 1·10 [0·87-1·40], p=0·62). BCG at birth inhibited the increase in histone trimethylation at the TNF promoter in peripheral blood mononuclear cells occurring in the first 6 weeks of life. H3K4me3 geometric mean fold-increases were 3·1 times lower at the TNF promoter (p=0·018), 2·5 times lower at the IL6 promoter (p=0·20), and 3·1 times lower at the IL1B promoter (p=0·082) and H3K9me3 geometric mean fold-increases were 8·9 times lower at the TNF promoter (p=0·0046), 1·2 times lower at the IL6 promoter (p=0·75), and 4·6 times lower at the IL1B promoter (p=0·068), in BCG-vaccinated (BCG at birth group) versus BCG-naive (delayed BCG group) infants. No clear effect of BCG on ex-vivo production of TNF, IL-6, IL-1β, IL-10, and IFNγ after heterologous stimulation, or transferrin saturation and hepcidin concentration, was detected (geometric mean ratios between 0·68 and 1·68; p≥0·038 for all comparisons).

Interpretation: BCG vaccination protects against non-tuberculous infectious disease during the neonatal period, in addition to having tuberculosis-specific effects. Prioritisation of BCG on the first day of life in high-mortality settings might have significant public-health benefits through reductions in all-cause infectious morbidity and mortality.

Funding: Wellcome Trust.

Translations: For the Luganda and Swahili translations of the abstract see Supplementary Materials section.
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http://dx.doi.org/10.1016/S1473-3099(20)30653-8DOI Listing
February 2021