Introduction:Necrotizing enterocolitis (NEC) is a leading cause of neonatal death and long-term morbidity, involving complex pathophysiology including prematurity, abnormal bacterial colonization, and ischemia-reperfusion injury, partially mediated by neutrophils. However, the limitations of current animal models hinder the development of targeted therapies for NEC. Thus, this study aimed to develop a human intestinal organoid model for NEC to investigate its pathophysiology, understand neutrophil involvement, and bridge animal and human research. Methods:Organoid cultures were established from human neonatal intestinal samples with NEC (n=7) and without gut inflammation (controls, n=7), treated with lipopolysaccharides (LPS), and/or cocultured with neutrophils. Flow cytometry quantified neutrophil survival (propidium iodide/Annexin-V), activation (CD11b/CD66b), and TLR-4 expression, as well as organoid TLR-4 expression and apoptosis markers. NEC status and neutrophil recruitment were analyzed using immunofluorescence. Results:After LPS administration, NEC organoids showed significantly increased TLR-4 expression, intestinal apoptosis markers, and NEC scores compared to controls, with more pronounced differences after neutrophil addition. Neutrophil activation markers were elevated when cocultured with both NEC and control organoids, but TLR-4 expression increased only with NEC organoids. Discussion:The findings suggest that epithelial cells from NEC patients have a heightened innate TLR-4 expression upon LPS stimulation, potentially contributing to NEC development. LPS stimulation resulted in more pronounced NEC-like lesions in NEC organoids, which were exacerbated by neutrophils. This model demonstrates that neutrophils might contribute to NEC manifestation and maintenance and that NEC organoids can reflect disease aspects, potentially aiding in the development of targeted therapies.

Globally, 16 million children are HIV-exposed but uninfected (HEU), with health disparities when compared with children who are HIV-unexposed. To identify current challenges, a research prioritisation exercise was conducted through an online survey and in-person workshop with diverse stakeholders, with the aim of identifying the top ten scientific priorities related to children who are HEU. Among 104 survey respondents (46% from Africa; 37% from Europe; 15% from the region of the Americas; and 2% from South-East Asia), 271 research questions were submitted. Workshop attendees (n=35) refined and prioritised questions through discussion and consensus. The highest research priority was identifying strategies to reduce morbidity and mortality and to improve growth and neurodevelopment among children who are HEU. Other research questions included: defining the underlying causes of health disparities; identifying clinically vulnerable mother-infant pairs through screening; documenting life course outcomes; defining the contribution of antiretrovirals to health disparities; identifying clinically important immune perturbations; understanding which infections should be prevented, and how; exploring intergenerational effects; defining meaningful health disparities; and characterising drivers of immune abnormalities. Participants also identified several methodological considerations and strategic approaches to advance the field. Coordinated action between communities, researchers, clinicians, policy makers, and funders is now required to conduct high-quality research to address these priority needs, with a particular focus on regions with high HIV prevalence.

BACKGROUND:Cell metabolism is critical for adequate responses by immune cells. Although changes in immune cell populations in uninfected children born to women living with HIV (HEU children) are well described, little is known regarding the metabolic characteristics of their immune cells. METHODS:Peripheral blood mononuclear cells of 110 children at approximately 6 years of age were analysed with flow cytometry to characterize natural killer (NK) cells and measure fatty acid uptake (BODIPY FL C 16 ) and mitochondrial mass [MitoTracker Green (MTG)]. HEU children born to women initiating antiretroviral therapy (ART) during pregnancy [medium-term ART exposure (HEUMT)] ( N  = 30), before conception [long-term ART exposure (HEULT)] ( N  = 39), and 41 HIV-unexposed uninfected (HUU) children were included. RESULTS:Overall, NK cell frequencies were similar between HEU and HUU children. However, NKG2A +/- CD56 dim NK cells in HEUMT children had a lower fatty acid (FA) uptake compared to HEULT and HUU children. NKG2A + CD57 - CD56 dim NK cells in HEULT children had a significantly higher mitochondrial mass compared to HEUMT and HUU children. Granzyme B and perforin negatively correlated with mean fluorescence intensity (MFI) MTG in NKG2A +/- CD56 dim NK cells in HEU and HUU children, but positively correlated with MFI BODIPY FL C 16 , in HEULT children, indicating that increased mitochondrial mass and lower FA acid uptake is associated with a lower cytotoxic potential. CONCLUSION:Taken together, mitochondrial mass and FA uptake differ between HEU and HUU children at 6 years of age suggesting long-term metabolic changes in HEU children. Further studies are needed to assess whether these have implications for immune competence in HEU children.

ABSTRACT High replicative capacity (RC) HIV-1 strains are associated with elevated viral loads and faster disease progression in the absence of antiretroviral therapy. Understanding the mechanisms by which high RC strains adversely affect the host is essential for developing novel anti-HIV interventions. This study investigates cellular metabolism, cytokine induction, and cell-to-cell spread as potential mechanisms differentiating clinical outcomes between low and high RC strains of HIV-1. We constructed chimeric viruses containing patient-derived gag-proteases from HIV-1 subtypes B and C in the NL4-3 backbone. Viral RC was determined using a green fluorescent protein (GFP)-reporter T-cell line assay and cytokine production in T-cells was assessed using Luminex. Virus cell-to-cell spread efficiency was measured through flow cytometry-based detection of p24, while nutrient uptake assays and mitotracker dye detection served as surrogate markers for T-cell metabolism and mitochondrial function. Chimeric subtype C viruses exhibited significantly lower RC compared to subtype B viruses (P = 0.0008). Cytokine profiling revealed distinct cytokine signatures associated with low RC subtype C viruses. Viral RC negatively correlated with tumor necrosis factor alpha (TNF-α), IL-8, and IL-13 induction, while it positively correlated with platelet-derived growth factor (PDGF-bb), IL-7, monocyte chemoattractant protein-1 (MCP-1), fibroblast growth factor (FGF)-basic levels, viral spread efficiency (P = 0.008, r = 0.5), and cellular glucose uptake (P = 0.02, r = 0.5). Conversely, RC was negatively correlated with glutamine levels (P = 0.001, r = −0.7), indicating a link between RC and nutrient utilization. Furthermore, mitochondrial depolarization was elevated in subtype B infections when compared to subtype C infections (P = 0.0008). These findings indicate that high RC strains exert distinct cellular effects that may influence HIV-1 pathogenesis, highlighting the need to develop novel therapeutic strategies.IMPORTANCEVirus replicative capacity (RC) influences disease progression following HIV-1 transmission; however, the mechanisms underlying the differential clinical outcomes remain poorly understood. Our study reveals variations in cytokine induction and cellular metabolism in T-cells infected with HIV-1 subtype B and C viruses exhibiting high or low RC. T-cells infected with high RC strains showed increased induction of IL-7 and platelet-derived growth factor (PDGF-bb), along with heightened glucose uptake and elevated glutamine consumption compared to those infected with low RC strains. By contrast, low RC strains induced higher levels of IL-8, IL-13, and tumor necrosis factor alpha (TNF-α) and demonstrated reduced efficiency in modulating cellular metabolism and virus cell-to-cell spreadability. These findings highlight distinct biological differences between high and low RC viruses, providing valuable insights into the mechanisms that may underpin varying clinical outcomes. This knowledge may inform the development of novel interventions aimed at limiting viral virulence or transmission.

Abstract Background HIV-exposed uninfected (HEU) children are at increased risk of morbidity during the first years of life. Although the immune responses of HEU infants in early-life are relatively well described, studies of natural killer (NK) cells in older HEU children are lacking. NK cell subsets were analysed in HEU children and compared to those in HIV unexposed uninfected (HUU) children aged ~ five years. Methods Multi-parametric flow cytometry was used to characterize peripheral blood-derived NK cell CD56, CD16, CD57, NKG2A and KIR3DL1/KIR2DL2/L3 expression, including intracellular perforin and granzyme B. NK cell subsets were compared between HEU children exposed to prenatal antiretroviral therapy (ART) from conception [long-term (HEULT)]; those exposed to ART during pregnancy [medium-term (HEUMT)] with continued exposure throughout the breastfeeding period and HUU peers. Furthermore, clinical data of the children, including sick clinic visits and hospitalizations documented in morbidity diaries from birth to 5 years were compared between HEU and HUU groups. Frequencies of CD56bright and CD56dim NK cell were correlated with these clinical parameters. Results 139 children were enrolled however, 133 comprising 43 HEULT, 38 HEUMT and 52 HUU were included in the main analyses. Total NK cell, CD56bright nor CD56dim NK cell proportions differed between HEU and HUU children. However, HEULT children had lower frequencies of CD56dim NK cells compared to HEUMT children, (p = 0.002) which maintained significance after controlling for preterm birth, p = 0.012. No differences were observed between HEULT and HUU. The expressions of NKG2A, KIR3DL1/KIR2DL2/L3 and CD57 on CD56bright and CD56dim NK cells were similar between the three groups. Furthermore, the frequencies of granzyme B and perforin double positive NK cells were similar between the HUU with HEULT and HEUMT children. CD56dim NK cell counts had a significant moderate negative correlation with recurrent respiratory infections (rho=-0.38; p = 0.010) in HUU children and negatively correlated with total sick clinic visits in HEUMT (rho=-0.40, p = 0.064). Conclusion The proportions of total NK cell, CD56bright and CD56dim NK cells, NK cells inhibitory and differentiation surface marker expression and cytolytic granule-positive cells were similar between HEU and HUU children. These data suggest that early-life HIV/ART exposure may not result in major changes in NK cell subsets at 5 years of age.

Gopee and colleagues’ recent analyses of diverse high-dimensional datasets of prenatal and adult skin, together with data from complex skin organoids, uncover the important contributions of macrophages in modulating prenatal skin development, scarless wound healing, and angiogenesis. These findings identify a role for skin immune cells in tissue development.

Human immunodeficiency virus type 1 (HIV-1) disease manifestations differ between cisgender women and men, including better control of viral replication during primary infection and less frequent residual HIV-1 replication on antiretroviral therapy (ART) in cisgender women with HIV-1 (WWH). Investigating plasmacytoid dendritic cell (pDC) functions and HIV-1 reservoir sizes in 20 WWH on stable ART, we observed inverse correlations between interferon-α and tumor necrosis factor responses of pDCs to Toll-like receptor 7/8 stimulation and intact/total proviral HIV-1 DNA levels. Additionally, ISG15 mRNA levels in peripheral blood mononuclear cells correlated with cytokine responses of pDCs. These findings demonstrate an association between higher type I interferon responses and lower HIV-1 reservoir sizes in WWH on ART, warranting studies to identify the underlying mechanisms.

IntroductionAppendicitis is one of the most common causes of acute abdominal surgery in children. The clinical course of appendicitis ranges from simple to complex appendicitis. The mechanisms underlying the heterogeneity of appendicitis in children remain largely unclear. Dysregulated T cell responses play an important role in several inflammatory diseases of the intestine, but the extend of T cell dysregulation in appendicitis in children is less well known.MethodsTo characterize appendiceal T cells in simple and complex appendicitis we performed in-depth immunophenotyping of appendiceal-derived T cells by flow cytometry and correlated this to appendiceal-derived microbiota analyses of the same patient.ResultsAppendix samples of twenty children with appendicitis (n = 8 simple, n = 12 complex) were collected. T cells in complex appendicitis displayed an increased differentiated phenotype compared to simple appendicitis, including a loss of both CD27 and CD28 by CD4+ T cells and to a lesser extent by CD8+ T cells. Frequencies of phenotypic tissue-resident memory CD69+CD4+ T cells and CD69+CD8+ T cells were decreased in children with complex compared to simple appendicitis, indicating disruption of local tissue-resident immune responses. In line with the increased differentiated phenotype, cytokine production of in particular IL-17A by CD4+ T cells was increased in children with complex compared to simple appendicitis. Furthermore, frequencies of IL-17A+ CD4+ T cells correlated with a dysregulation of the appendiceal microbiota in children with complex appendicitis.ConclusionIn conclusion, disruption of local T cell responses, and enhanced pro-inflammatory Th17 responses correlating to changes in the appendiceal microbiota were observed in children with complex compared to simple appendicitis. Further studies are needed to decipher the role of a dysregulated network of microbiota and Th17 cells in the development of complex appendicitis in children.

Recent progress in flow and mass cytometry technologies enables the simultaneous measurement of over 50 parameters for an individual cell. The resulting increase in data volume and complexity present challenges, as conventional analysis methods based on manual gating are time-consuming and fail to capture unknown or minor cell populations. Advances in single-cell RNA sequencing (scRNAseq) technologies have prompted the development of sophisticated computational analysis tools specifically designed to process and analyze high-dimensional biological data, some of which could significantly improve certain aspects of cytometry data analysis. Building on these advances, we here present Seumetry, a framework that combines flow and mass cytometry data-specific analysis methods with the capabilities of Seurat, a powerful tool for the analysis of scRNAseq data. Seumetry offers advanced quality control, data visualizations, and differential population abundance and protein expression analysis. We tested Seumetry on an in-house generated complex dataset of immune cells from different layers of human intestines, demonstrating that Seumetry accurately identifies distinct immune cell populations. Furthermore, using a publicly available mass cytometry dataset, Seumetry recapitulates previously published results, further validating its use for high-dimensional flow and mass cytometry data. In summary, Seumetry provides a new scalable framework for the comprehensive analysis of high-dimensional cytometry data with seamless integration into commonly used scRNAseq analysis tools, enabling in-depth analysis methods to facilitate biological interpretations. ### Competing Interest Statement The authors have declared no competing interest.

Objective Primary sclerosing cholangitis (PSC) is characterised by bile duct strictures and progressive liver disease, eventually requiring liver transplantation. Although the pathogenesis of PSC remains incompletely understood, strong associations with HLA-class II haplotypes have been described. As specific HLA-DP molecules can bind the activating NK-cell receptor NKp44, we investigated the role of HLA-DP/ NKp44-interactions in PSC. Design Liver tissue, intrahepatic and peripheral blood lymphocytes of individuals with PSC and control individuals were characterised using flow cytometry, immunohistochemical and immunofluorescence analyses. HLA-DPA1 and HLA-DPB1 imputation and association analyses were performed in 3408 individuals with PSC and 34 213 controls. NK cell activation on NKp44/HLA-DP interactions was assessed in vitro using plate-bound HLA-DP molecules and HLA-DPB wildtype versus knock-out human cholangiocyte organoids. Results NKp44+NK cells were enriched in livers, and intrahepatic bile ducts of individuals with PSC showed higher expression of HLA-DP. HLA-DP haplotype analysis revealed a highly elevated PSC risk for HLA-DPA1* 02:01 similar to B1*01:01 (OR 1.99, p=6.7x10-50). Primary NKp44+NK cells exhibited significantly higher degranulation in response to plate-bound HLA-DPA1* 02:01-DPB1* 01:01 compared with control HLA-DP molecules, which were inhibited by anti-NKp44-blocking. Human cholangiocyte organoids expressing HLA-DPA1* 02:01-DPB1* 01:01 after IFN-gamma-exposure demonstrated significantly increased binding to NKp44-Fc constructs compared with unstimulated controls. Importantly, HLA-DPA1* 02:01-DPB1* 01:01-expressing organoids increased degranulation of NKp44+NK cells compared with HLA-DPB1- KO organoids. Conclusion Our studies identify a novel PSC risk haplotype HLA-DP A1*02:01 similar to DPB1*01:01 and provide clinical and functional data implicating NKp44+NK cells that recognise HLA-DPA1* 02:01-DPB1* 01:01 expressed on cholangiocytes in PSC pathogenesis.

Background. Immune dysfunction often persists in people living with human immunodeficiency virus (HIV) who are on antiretroviral therapy (ART), clinically manifesting as HIV-1-associated comorbid conditions. Early ART initiation may reduce incidence of HIV-1-associated immune dysfunction and comorbid conditions. Immunometabolism is a critical determinant of functional immunity. We investigated the effect of HIV-1 infection and timing of ART initiation on CD4(+) T cell metabolism and function.Methods. Longitudinal blood samples from people living with HIV who initiated ART during hyperacute HIV-1 infection (HHI; before peak viremia) or chronic HIV-1 infection (CHI) were assessed for the metabolic and immune functions of CD4(+) T cells. Metabolite uptake and mitochondrial mass were measured using fluorescent analogues and MitoTracker Green accumulation, respectively, and were correlated with CD4(+) T cell effector functions.Results. Initiation of ART during HHI prevented dysregulation of glucose uptake by CD4(+) T cells, but glucose uptake was reduced before and after ART initiation in CHI. Glucose uptake positively correlated with interleukin-2 and tumor necrosis factor-alpha production by CD4(+) T cells. CHI was associated with elevated mitochondrial mass in effector memory CD4(+) T cells that persisted after ART and correlated with PD-1 expression.Conclusions. ART initiation in HHI largely prevented metabolic impairment of CD4(+) T cells. ART initiation in CHI was associated with persistently dysregulated immunometabolism of CD4(+) T cells, which was associated with impaired cellular functions and exhaustion.

Although natural killer (NK) cells are recognized for their modulation of immune responses, the mechanisms by which human NK cells mediate immune regulation are unclear. Here, we report that expression of human leukocyte antigen (HLA)-DP, a ligand for the activating NK cell receptor NKp44, is significantly upregulated on CD8+ effector T cells, in particular in human cytomegalovirus (HCMV)+ individuals. HLA-DP+ CD8+ T cells expressing NKp44-binding HLA-DP antigens activate NKp44+ NK cells, while HLA-DP+ CD8+ T cells not expressing NKp44-binding HLA-DP antigens do not. In line with this, frequencies of HLA-DP+ CD8+ T cells are increased in individuals not encoding for NKp44-binding HLA-DP haplotypes, and contain hyper-expanded CD8+ T cell clones, compared to individuals expressing NKp44-binding HLA-DP molecules. These findings identify a molecular interaction facilitating the HLA-DP haplotype-specific editing of HLA-DP+ CD8+ T cell effector populations by NKp44+ NK cells and preventing the generation of hyper-expanded T cell clones, which have been suggested to have increased potential for autoimmunity.

The crosstalk between NK cells and their surrounding environment is enabled through activating and inhibitory receptors, which tightly control NK cell activity. The co-inhibitory receptor TIGIT decreases NK cell cytotoxicity and is involved in NK cell exhaustion, but has also been associated with liver regeneration, highlighting that the contribution of human intrahepatic CD56 bright NK cells in regulating tissue homeostasis remains incompletely understood. A targeted single-cell mRNA analysis revealed distinct transcriptional differences between matched human peripheral blood and intrahepatic CD56 bright NK cells. Multiparameter flow cytometry identified a cluster of intrahepatic NK cells with overlapping high expression of CD56, CD69, CXCR6, TIGIT and CD96. Intrahepatic CD56 bright NK cells also expressed significantly higher protein surface levels of TIGIT, and significantly lower levels of DNAM-1 compared to matched peripheral blood CD56 bright NK cells. TIGIT + CD56 bright NK cells showed diminished degranulation and TNF-α production following stimulation. Co-incubation of peripheral blood CD56 bright NK cells with human hepatoma cells or primary human hepatocyte organoids resulted in migration of NK cells into hepatocyte organoids and upregulation of TIGIT and downregulation of DNAM-1 expression, in line with the phenotype of intrahepatic CD56 bright NK cells. Intrahepatic CD56 bright NK cells represent a transcriptionally, phenotypically, and functionally distinct population of NK cells that expresses higher levels of TIGIT and lower levels of DNAM-1 than matched peripheral blood CD56 bright NK cells. Increased expression of inhibitory receptors by NK cells within the liver environment can contribute to tissue homeostasis and reduction of liver inflammation.

Adenine nucleotides (AN) are ubiquitous metabolites that regulate cellular energy metabolism and modulate cell communication and inflammation. To understand how disturbances in AN balance arise and affect cellular function, robust quantification techniques for these metabolites are crucial. However, due to their hydrophilicity, simultaneous quantification of AN across various biological samples has been challenging. Here we present a hydrophilic interaction high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) based method for the quantification of 26 adenosine nucleotides and precursors as well as metabolic products of nicotinamide adenine dinucleotide (NAD) in plasma, liver, and adipose tissue samples as well as cell culture supernatants and cells. Method validation was performed with regard to linearity, accuracy, precision, matrix effects, and carryover. Finally, analysis of cell culture supernatants derived from intestinal organoids and RAW 264.7 cells illustrates that the here described method is a reliable and easy-to-use tool to quantify AN and opens up new avenues to understand the role of AN generation and breakdown for cellular functions.

BACKGROUND & AIMS: Ulcerative colitis (UC) is characterized by severe inflammation and destruction of the intestinal epithelium, and is associated with specific risk single nucleotide polymorphisms in HLA class II. Given the recently discovered interactions between subsets of HLA-DP molecules and the activating natural killer (NK) cell receptor NKp44, genetic as-sociations of UC and HLA-DP haplotypes and their functional implications were investigated. METHODS: HLA-DP haplotype and UC risk association analyses were performed (UC: n = 13,927; control: n = 26,764). Expression levels of HLA-DP on intestinal epithelial cells (IECs) in individuals with and without UC were quantified. Human intestinal 3-dimensional (3D) organoid cocultures with human NK cells were used to deter-mine functional consequences of interactions between HLA-DP and NKp44. RESULTS: These studies identified HLA-DPA1*01:03-DPB1*04:01 (HLA-DP401) as a risk haplotype and HLA-DPA1*01:03-DPB1*03:01 (HLA-DP301) as a protective haplotype for UC in European populations. HLA-DP expression was significantly higher on IECs of individuals with UC compared with controls. IECs in human intestinal 3D organoids derived from HLA-DP401pos individuals showed significantly stronger binding of NKp44 compared with HLA-DP301pos IECs. HLA-DP401pos IECs in organoids triggered increased degranu-lation and tumor necrosis factor production by NKp44+ NK cells in cocultures, resulting in enhanced epithelial cell death compared with HLA-DP301pos organoids. Blocking of HLA-DP401-NKp44 interactions (anti-NKp44) abrogated NK cell activity in cocultures. CONCLUSIONS: We identified an UC risk HLA-DP haplotype that engages NKp44 and activates NKp44+ NK cells, mediating damage to intestinal epithelial cells in an HLA-DP haplotype-dependent manner. The molecular interac-tion between NKp44 and HLA-DP401 in UC can be targeted by therapeutic interventions to reduce NKp44+ NK cell-mediated destruction of the intestinal epithelium in UC.

Type I interferons (IFN-I) are important mediators of antiviral immunity and autoimmune diseases. Female plasmacytoid dendritic cells (pDCs) exert an elevated capacity to produce IFN-I upon toll-like receptor 7 (TLR7) activation compared to male pDCs, and both sex hormones and X-encoded genes have been impli-cated in these sex-specific differences. Using longitudinal samples from a trans men cohort receiving gender-affirming hormone therapy (GAHT), the impact of testosterone injections on TLR7-mediated IFN-I production by pDCs was assessed. Single-cell RNA analyses of pDCs showed downregulation of IFN-I-related gene expression signatures but also revealed transcriptional inter-donor heterogeneity. Longitudinal quantification showed continuous reduction of IFN-I protein production by pDCs and reduced expression of IFN-I-stimulated genes in peripheral blood mononuclear cells (PBMCs). These studies in trans men demonstrate that testosterone administration reduces IFN-I production by pDCs over time and provide insights into the immune-modulatory role of testosterone in sex-specific IFN-I-mediated immune responses.

Commencing lifelong antiretroviral therapy (ART) immediately following HIV diagnosis (Option B+) has dramatically improved the health of HIV-infected women and their children, with the majority being of HIV-exposed children born uninfected (HEU). This success has led to an increasing population of HIV-infected women receiving ART during pregnancy and children exposed to ART . Nonetheless, a small proportion of children are still infected with HIV (HEI) each year. HEI children suffer from reduced immunocompetence and host-defence, due to CD4+ T lymphocyte depletion, but also dysregulation of other immune cells including CD8+ T lymphocytes, natural killer (NK) cells, macrophages including B lymphocytes. Furthermore, although HEU children are uninfected, altered immune responses are observed and associated with increased vulnerability to infections. The mechanisms underlying immune dysregulation in HEU children remain poorly described. Building on early studies, emerging data suggests that HIV/ART exposure early in life affects cell metabolic function of HEU children. Prenatal HIV/ART exposure has been associated with dysregulation of mitochondria, including impaired DNA polymerase activity. Furthermore, dysregulation of oxidative phosphorylation (OXPHOS) causes a decreased generation of adenosine triphosphate (ATP) and increased production of reactive oxygen species (ROS), resulting in oxidative stress. These altered metabolic processes can affect immune cell viability and immune responses. Recent studies have indicated that immune-metabolic dysregulation may contribute to HIV-associated pathogenesis and clinical observations associated with HIV and ART exposure in HEU/HEI children. Given the critical role metabolic processes in immune cell functioning, immune-metabolic dysregulation in HEU and HEI children may have implications in effective host-defence responses against pathogens, as well as efficacy of standard ART regimens and future novel HIV cure approaches in HEI children. At the same time, targeting metabolic pathways of immune cells may provide safer and novel approaches for HIV cure strategies. Here, we review the current literature investigating immune-metabolic dysregulation in paediatric HIV pathogenesis.

Early life is characterized by extraordinary challenges, including rapid tissue growth and immune adaptation to foreign antigens after birth. During this developmental stage, infants have an increased risk of immune-mediated diseases. Here, we demonstrate that tissue-resident, interleukin (IL)-13- and IL-4-producing group 2 innate lymphoid cells (ILC2s) are enriched in human infant intestines compared to adult intestines. Organoid systems were employed to assess the role of infant intestinal ILC2s in intestinal development and showed that IL-13 and IL-4 increased epithelial cell proliferation and skewed cell differentiation toward secretory cells. IL-13 furthermore upregulated the production of mediators of type-2 immunity by infant intestinal epithelial cells, including vascular endothelial growth factor-A and IL-26, a chemoattractant for eosinophils. In line with these in vitro findings increased numbers of eosinophils were detected in vivo in infant intestines. Taken together, ILC2s are enriched in infant intestines and can support intestinal development while inducing an epithelial secretory response associated with type 2 immune-mediated diseases.

The intestine is constantly balancing the maintenance of a homeostatic microbiome and the protection of the host against pathogens such as viruses. Many cytokines mediate protective inflammatory responses in the intestine, among them IL-1β. IL-1β is a proinflammatory cytokine typically activated upon specific danger signals sensed by the inflammasome. SARS-CoV-2 is capable of infecting multiple organs, including the intestinal tract. Severe cases of COVID-19 were shown to be associated with a dysregulated immune response, and blocking of proinflammatory pathways was demonstrated to improve patient survival. Indeed, anakinra, an Ab against the receptor of IL-1β, has recently been approved to treat patients with severe COVID-19. However, the role of IL-1β during intestinal SARS-CoV-2 infection has not yet been investigated. Here, we analyzed postmortem intestinal and blood samples from patients who died of COVID-19. We demonstrated that high levels of intestinal IL-1β were associated with longer survival time and lower intestinal SARS-CoV-2 RNA loads. Concurrently, type I IFN expression positively correlated with IL-1β levels in the intestine. Using human intestinal organoids, we showed that autocrine IL-1β sustains RNA expression of IFN type I by the intestinal epithelial layer. These results outline a previously unrecognized key role of intestinal IL-1β during SARS-CoV-2 infection.

Objective: To determine immune-metabolic dysregulation in children born to women living with HIV. Methods: Longitudinal immune-metabolomic analyses of plasma of 32 pregnant women with HIV (WHIV) and 12 uninfected women and their children up to 1.5 years of age were performed. Results: Using liquid chromatography-mass spectrometry and a multiplex bead assay, 280 metabolites (57 amino acids, 116 positive lipids, 107 signalling lipids) and 24 immune mediators (e.g. cytokines) were quantified. combinational antiretroviral therapy (cART) exposure was categorized as cART initiation preconception (long), cART initiation postconception up to 4 weeks before birth (medium) and cART initiation within 3 weeks of birth (short). Plasma metabolite profiles differed between HIV-exposed-uninfected (HEU)-children with long cART exposure compared to HIV-unexposed-children (HUU). Specifically, higher levels of methionine-sulfone, which is associated with oxidative stress, were detected in HEU-children with long cART exposure compared to HUU-children. High infant methionine-sulfone levels were reflected by high prenatal plasma levels in the mother. Increased methionine-sulfone levels in the children were associated with decreased growth, including both weight and length. Conclusion: These findings based on longitudinal data demonstrate that dysregulation of metabolite networks associated with oxidative stress in children born to WHIV is associated with restricted infant growth.