Graham A. Heieis

Intestinal dendritic cells (DCs) play a central role in maintaining gut tolerance through priming of peripheral regulatory T cells (pT _reg cells). DC tolerogenicity has been linked to catabolic metabolism, but the role of AMP-activated kinase (AMPK), a key regulator of catabolic metabolism, in regulating intestinal tolerance remains unclear. We found high AMPK activation in intestinal DCs, and loss of AMPKα1 in CD11c-expressing cells (CD11c ^ΔAMPKα1 ) led to reduced frequencies of intestinal RALDH ⁺ CD103 ⁺ cDC2s. This was associated with reduced pT _reg cell induction and consequently increased type 2 immunity in models of intestinal helminth infection. Correspondingly, CD103 ⁺ cDC2s from helminth-infected CD11c ^ΔAMPKα1 mice failed to prime T _reg cells ex vivo. Similarly, T _reg cell induction by AMPK-deficient human retinoic acid (RA)–induced tolerogenic CD103 ⁺ DCs was compromised. Mechanistically, AMPK underpinned RA-driven tolerogenicity by promoting RALDH activity and TGF-β secretion in a FoxO3-dependent manner, independent from metabolic reprogramming. Our findings identify AMPK as a key regulator of intestinal DC-mediated tolerance and as a therapeutic target to counter intestinal inflammatory disease.

Immune cell activity is strongly influenced by the nutrients present during activation. The effects of specific fatty acids (FAs) can be particularly complex, with them exerting diverse and sometimes opposing effects on immune cells. These functional differences are thought to stem from structural differences between the FAs, leading to altered cellular handling. However, chemical tools to directly probe these aspects remain limited. Here, we report the design and synthesis of saturated, unsaturated, and polyunsaturated cyclopropenyl fatty acids, each incorporating a minimal one‐carbon cyclopropene moiety as a bioorthogonal click handle. This motif enables rapid and live‐cell‐compatible labeling via the inverse electron‐demand Diels–Alder reaction, providing a versatile platform to trace FA behavior in biological systems. Application of these synthetic cyclopropenyl FAs in primary immune cell mixtures reveals distinct uptake patterns between different cell types, with polyunsaturated analogues showing strong uptake across all immune cell types. Complementary metabolic and proteomic analyses suggest that this uptake results in biological differences between high/low uptake immune populations, highlighting the utility of cyclopropenyl FA probes for dissecting lipid uptake in the context of immune cell biology.

Immune cell activity is strongly influenced by the nutrients present during activation. The effects of specific fatty acids (FAs) can be particularly complex, with them exerting diverse and sometimes opposing effects on immune cells. These functional differences are thought to stem from structural differences between the FAs, leading to altered cellular handling. However, chemical tools to directly probe these aspects remain limited. Here, we report the design and synthesis of saturated, unsaturated, and polyunsaturated cyclopropenyl fatty acids, each incorporating a minimal one‐carbon cyclopropene moiety as a bioorthogonal click handle. This motif enables rapid and live‐cell‐compatible labeling via the inverse electron‐demand Diels–Alder reaction, providing a versatile platform to trace FA behavior in biological systems. Application of these synthetic cyclopropenyl FAs in primary immune cell mixtures reveals distinct uptake patterns between different cell types, with polyunsaturated analogues showing strong uptake across all immune cell types. Complementary metabolic and proteomic analyses suggest that this uptake results in biological differences between high/low uptake immune populations, highlighting the utility of cyclopropenyl FA probes for dissecting lipid uptake in the context of immune cell biology.

Automated transmission electron microscopy (TEM) generates large datasets that challenge traditional qualitative analysis of cellular ultrastructure. Quantitative assessment of structural differences between different samples remains difficult due to structural variability in thin sections of organelles. Here we applied supervised machine learning (sML) to segment, quantify and compare cellular structures -including nuclei, chromatin, mitochondria, rough endoplasmic reticulum, and endocytic vesicles- in large TEM images of wild-type macrophages versus those with altered cellular physiology due to deficiency in O-GlcNAc Transferase (OGT). sML revealed that OGT knockout macrophages are larger and more oval, with increased euchromatin, nucleoli size, and relative mitochondrial and rER surface areas. Comparison with six TEM experts showed sML provides more objective and sensitive quantification of subtle differences, while expert consensus is only achieved for larger structural variations. These findings demonstrate that sML enhances quantitative TEM analysis and complements human expertise in ultrastructural studies.

Urbanization affects environmental exposures and lifestyle, shaping immune system variation and influencing disease susceptibility and vaccine responses. Here, we present systems analysis of immune profiles across the rural-urban gradient, comparing rural and urban Senegalese with urban Dutch individuals. By integrating single-cell phenotyping, metabolic profiling, and functional analysis, we reveal a trajectory of immune remodeling along the gradient. This includes enrichment of proinflammatory CD11c ⁺ B cells associated with altered IgG Fc glycosylation, adaptive NK cells with reduced responsiveness to accessory cytokines, and CD161 ⁺ CD4 ⁺ T cells with enhanced cytokine production in rural settings. Metabolic perturbation studies demonstrated distinct dependencies on glycolysis, pentose phosphate pathway, and fatty acid synthesis for cellular cytokine responses across populations. We validate core rural-urban immune signatures in an independent Indonesian cohort, suggesting shared immunological adaptations to urbanization across ancestries and geographical areas. Our findings provide insights into rural-urban immune function in understudied populations.

Cytomegalovirus (CMV) is one of the most common and relevant opportunistic pathogens in people who are immunocompromised, such as kidney transplant recipients (KTRs). The exact mechanisms underlying the disability of cytotoxic T cells to provide sufficient protection against CMV in people who are immunosuppressed have not been identified yet. Here, we performed in-depth metabolic profiling of CMV-specific CD8 ⁺ T cells in patients who are immunocompromised and show the development of metabolic dysregulation at the transcriptional, protein, and functional level of CMV-specific CD8 ⁺ T cells in KTRs with noncontrolled CMV infection. These dysregulations comprise impaired glycolysis and increased mitochondrial stress, which is associated with an intensified expression of the nicotinamide adenine dinucleotide nucleotidase (NADase) CD38. Inhibiting NADase activity of CD38 reinvigorated the metabolism and improved cytokine production of CMV-specific CD8 ⁺ T cells. These findings were corroborated in a mouse model of CMV infection under conditions of immunosuppression. Thus, dysregulated metabolic states of CD8 ⁺ T cells could be targeted by inhibiting CD38 to reverse hyporesponsiveness in individuals who fail to control chronic viral infection.

Cellular metabolism is a key determinant of immune cell function. Here we found that CD14⁺ monocytes from Sub‐Saharan Africans produce higher levels of IL‐10 following TLR‐4 stimulation and are bioenergetically distinct from monocytes from Europeans. Through metabolomic profiling, we identified the higher IL‐10 production to be driven by increased baseline production of NADPH oxidase‐dependent reactive oxygen species, supported by enhanced pentose phosphate pathway activity. Together, these data indicate that NADPH oxidase‐derived ROS is a metabolic checkpoint in monocytes that governs their inflammatory profile and uncovers a metabolic basis for immunological differences across geographically distinct populations.

Tissue-resident macrophage populations constitute a mosaic of phenotypes, yet how their metabolic states link to the range of phenotypes and functions in vivo is still poorly defined. Here, using high-dimensional spectral flow cytometry, we observe distinct metabolic profiles between different organs and functionally link acetyl CoA carboxylase activity to efferocytotic capacity. Additionally, differences in metabolism are evident within populations from a specific site, corresponding to relative stages of macrophage maturity. Immune perturbation with intestinal helminth infection increases alternative activation and metabolic rewiring of monocyte-derived macrophage populations, while resident TIM4⁺ intestinal macrophages remain immunologically and metabolically hyporesponsive. Similar metabolic signatures in alternatively-activated macrophages are seen from different tissues using additional helminth models, but to different magnitudes, indicating further tissue-specific contributions to metabolic states. Thus, our high-dimensional, flow-based metabolic analyses indicates complex metabolic heterogeneity and dynamics of tissue-resident macrophage populations at homeostasis and during helminth infection.