The AMPK activator ATX-304 alters cellular metabolism to protect against cisplatin-induced acute kidney injury.

Acute kidney injury (AKI) disrupts energy metabolism. Targeting metabolism through AMP-activated protein kinase (AMPK) may alleviate AKI. ATX-304, a pan-AMPK activator, was evaluated in C57Bl/6 mice and tubular epithelial cell (TEC) cultures. Mice received ATX-304 (1 mg/g) or control chow for 7 days before cisplatin-induced AKI (CI-AKI). Primary cultures of tubular epithelial cells (TECs) were pre-treated with ATX-304 (20 µM, 4 h) prior to exposure to cisplatin (20 µM, 23 h). ATX-304 increased acetyl-CoA carboxylase phosphorylation, indicating AMPK activation. It protected against CI-AKI measured by serum creatinine (control 0.05 + 0.03 mM vs ATX-304 0.02 + 0.01 mM, P = 0.03), western blot for neutrophil gelatinase-associated lipocalin (NGAL) (control 3.3 + 1.8-fold vs ATX-304 1.2 + 0.55-fold, P = 0.002), and histological injury (control 3.5 + 0.59 vs ATX-304 2.7 + 0.74, P = 0.03). In TECs, pre-treatment with ATX-304 protected against cisplatin-mediated injury, as measured by lactate dehydrogenase release, MTS cell viability, and cleaved caspase 3 expression. ATX-304 protection against cisplatin was lost in AMPK-null murine embryonic fibroblasts. Metabolomic analysis in TECs revealed that ATX-304 (20 µM, 4 h) altered 66/126 metabolites, including fatty acids, tricarboxylic acid cycle metabolites, and amino acids. Metabolic studies of live cells using the XFe96 Seahorse analyzer revealed that ATX-304 increased the basal TEC oxygen consumption rate by 38%, whereas maximal respiration was unchanged. Thus, ATX-304 protects against cisplatin-mediated kidney injury via AMPK-dependent metabolic reprogramming, revealing a promising therapeutic strategy for AKI.

Inhibition of MERTK reduces organ fibrosis in mouse models of fibrotic disease.

Transforming growth factor-ß (TGFß) drives fibrosis and disease progression in a number of chronic disorders, but targeting this ubiquitously expressed cytokine may not yield a viable and safe antifibrotic therapy. Here, we sought to identify alternative ways to inhibit TGFß signaling using human hepatic stellate cells and macrophages from humans and mice in vitro, as well as mouse models of liver, kidney, and lung fibrosis. We identified Mer tyrosine kinase (MERTK) as a TGFß-inducible effector of fibrosis that was up-regulated during fibrosis in multiple organs in three mouse models. We confirmed these findings in liver biopsy samples from patients with metabolic dysfunction-associated fatty liver disease (MAFLD). MERTK also induced TGFß expression and drove TGFß signaling resulting in a positive feedback loop that promoted fibrosis in cultured cells. MERTK regulated both canonical and noncanonical TGFß signaling in both mouse and human cells in vitro. MERTK increased transcription of genes regulating fibrosis by modulating chromatin accessibility and RNA polymerase II activity. In each of the three mouse models, disrupting the fibrosis-promoting signaling loop by reducing MERTK expression reduced organ fibrosis. Pharmacological inhibition of MERTK reduced fibrosis in these mouse models either when initiated immediately after injury or when initiated after fibrosis was established. Together, these data suggest that MERTK plays a role in modulating organ fibrosis and may be a potential target for treating fibrotic diseases.

ß-cell function is regulated by metabolic and epigenetic programming of islet-associated macrophages, involving Axl, Mertk, and TGFß receptor signaling.

We have exploited islet-associated macrophages (IAMs) as a model of resident macrophage function, focusing on more physiological conditions than the commonly used extremes of M1 (inflammation) versus M2 (tissue remodeling) polarization. Under steady state, murine IAMs are metabolically poised between aerobic glycolysis and oxidative phosphorylation, and thereby exert a brake on glucose-stimulated insulin secretion (GSIS). This is underpinned by epigenetic remodeling via the metabolically regulated histone demethylase Kdm5a. Conversely, GSIS is enhanced by engaging Axl receptors on IAMs, or by augmenting their oxidation of glucose. Following high-fat feeding, efferocytosis is stimulated in IAMs in conjunction with Mertk and TGFß receptor signaling. This impairs GSIS and potentially contributes to ß-cell failure in pre-diabetes. Thus, IAMs serve as relays in many more settings than currently appreciated, fine-tuning insulin secretion in response to dynamic changes in the external environment. Intervening in this nexus might represent a means of preserving ß-cell function during metabolic disease.

Synonymous alterations of cancer-associated Trp53 CpG mutational hotspots cause fatal developmental jaw malocclusions but no tumors in knock-in mice.

Intragenic CpG dinucleotides are tightly conserved in evolution yet are also vulnerable to methylation-dependent mutation, raising the question as to why these functionally critical sites have not been deselected by more stable coding sequences. We previously showed in cell lines that altered exonic CpG methylation can modify promoter start sites, and hence protein isoform expression, for the human TP53 tumor suppressor gene. Here we extend this work to the in vivo setting by testing whether synonymous germline modifications of exonic CpG sites affect murine development, fertility, longevity, or cancer incidence. We substituted the DNA-binding exons 5-8 of Trp53, the mouse ortholog of human TP53, with variant-CpG (either CpG-depleted or -enriched) sequences predicted to encode the normal p53 amino acid sequence; a control construct was also created in which all non-CpG sites were synonymously substituted. Homozygous Trp53-null mice were the only genotype to develop tumors. Mice with variant-CpG Trp53 sequences remained tumor-free, but were uniquely prone to dental anomalies causing jaw malocclusion (p < .0001). Since the latter phenotype also characterises murine Rett syndrome due to dysfunction of the trans-repressive MeCP2 methyl-CpG-binding protein, we hypothesise that CpG sites may exert non-coding phenotypic effects via pre-translational cis-interactions of 5-methylcytosine with methyl-binding proteins which regulate mRNA transcript initiation, expression or splicing, although direct effects on mRNA structure or translation are also possible.

Apoptotic cell fragments locally activate tingible body macrophages in the germinal center.

Germinal centers (GCs) that form within lymphoid follicles during antibody responses are sites of massive cell death. Tingible body macrophages (TBMs) are tasked with apoptotic cell clearance to prevent secondary necrosis and autoimmune activation by intracellular self antigens. We show by multiple redundant and complementary methods that TBMs derive from a lymph node-resident, CD169-lineage, CSF1R-blockade-resistant precursor that is prepositioned in the follicle. Non-migratory TBMs use cytoplasmic processes to chase and capture migrating dead cell fragments using a "lazy" search strategy. Follicular macrophages activated by the presence of nearby apoptotic cells can mature into TBMs in the absence of GCs. Single-cell transcriptomics identified a TBM cell cluster in immunized lymph nodes which upregulated genes involved in apoptotic cell clearance. Thus, apoptotic B cells in early GCs trigger activation and maturation of follicular macrophages into classical TBMs to clear apoptotic debris and prevent antibody-mediated autoimmune diseases.

CARD11 gain-of-function mutation drives cell-autonomous accumulation of PD-1+ ICOShigh activated T cells, T-follicular, T-regulatory and T-follicular regulatory cells.

Introduction: Germline CARD11 gain-of-function (GOF) mutations cause B cell Expansion with NF-κB and T cell Anergy (BENTA) disease, whilst somatic GOF CARD11 mutations recur in diffuse large B cell lymphoma (DLBCL) and in up to 30% of the peripheral T cell lymphomas (PTCL) adult T cell leukemia/lymphoma (ATL), cutaneous T cell lymphoma (CTCL) and Sezary Syndrome. Despite their frequent acquisition by PTCL, the T cell-intrinsic effects of CARD11 GOF mutations are poorly understood. Methods: Here, we studied B and T lymphocytes in mice with a germline Nethyl-N-nitrosourea (ENU)-induced Card11M365K mutation identical to a mutation identified in DLBCL and modifying a conserved region of the CARD11 coiled-coil domain recurrently mutated in DLBCL and PTCL. Results and discussion: Our results demonstrate that CARD11.M365K is a GOF protein that increases B and T lymphocyte activation and proliferation following antigen receptor stimulation. Germline Card11M365K mutation was insufficient alone to cause B or T-lymphoma, but increased accumulation of germinal center (GC) B cells in unimmunized and immunized mice. Card11M365K mutation caused cell-intrinsic over-accumulation of activated T cells, T regulatory (TREG), T follicular (TFH) and T follicular regulatory (TFR) cells expressing increased levels of ICOS, CTLA-4 and PD-1 checkpoint molecules. Our results reveal CARD11 as an important, cell-autonomous positive regulator of TFH, TREG and TFR cells. They highlight T cell-intrinsic effects of a GOF mutation in the CARD11 gene, which is recurrently mutated in T cell malignancies that are often aggressive and associated with variable clinical outcomes.

Human PIK3R1 mutations disrupt lymphocyte differentiation to cause activated PI3Kδ syndrome 2.

Heterozygous loss-of-function (LOF) mutations in PIK3R1 (encoding phosphatidylinositol 3-kinase [PI3K] regulatory subunits) cause activated PI3Kδ syndrome 2 (APDS2), which has a similar clinical profile to APDS1, caused by heterozygous gain-of-function (GOF) mutations in PIK3CD (encoding the PI3K p110δ catalytic subunit). While several studies have established how PIK3CD GOF leads to immune dysregulation, less is known about how PIK3R1 LOF mutations alter cellular function. By studying a novel CRISPR/Cas9 mouse model and patients' immune cells, we determined how PIK3R1 LOF alters cellular function. We observed some overlap in cellular defects in APDS1 and APDS2, including decreased intrinsic B cell class switching and defective Tfh cell function. However, we also identified unique APDS2 phenotypes including defective expansion and affinity maturation of Pik3r1 LOF B cells following immunization, and decreased survival of Pik3r1 LOF pups. Further, we observed clear differences in the way Pik3r1 LOF and Pik3cd GOF altered signaling. Together these results demonstrate crucial differences between these two genetic etiologies.

Skin γδ T cell inflammatory responses are hardwired in the thymus by oxysterol sensing via GPR183 and calibrated by dietary cholesterol.

Dietary components and metabolites have a profound impact on immunity and inflammation. Here, we investigated how sensing of cholesterol metabolite oxysterols by γδ T cells impacts their tissue residency and function. We show that dermal IL-17-producing γδ T (Tγδ17) cells essential for skin-barrier homeostasis require oxysterols sensing through G protein receptor 183 (GPR183) for their development and inflammatory responses. Single-cell transcriptomics and murine reporter strains revealed that GPR183 on developing γδ thymocytes is needed for their maturation by sensing medullary thymic epithelial-cell-derived oxysterols. In the skin, basal keratinocytes expressing the oxysterol enzyme cholesterol 25-hydroxylase (CH25H) maintain dermal Tγδ17 cells. Diet-driven increases in oxysterols exacerbate Tγδ17-cell-mediated psoriatic inflammation, dependent on GPR183 on γδ T cells. Hence, cholesterol-derived oxysterols control spatially distinct but biologically linked processes of thymic education and peripheral function of dermal T cells, implicating diet as a focal parameter of dermal Tγδ17 cells.

An epithelial cell-derived metabolite tunes immunoglobulin A secretion by gut-resident plasma cells.

Immunoglobulin A (IgA) secretion by plasma cells, terminally differentiated B cells residing in the intestinal lamina propria, assures microbiome homeostasis and protects the host against enteric infections. Exposure to diet-derived and commensal-derived signals provides immune cells with organizing cues that instruct their effector function and dynamically shape intestinal immune responses at the mucosal barrier. Recent data have described metabolic and microbial inputs controlling T cell and innate lymphoid cell activation in the gut; however, whether IgA-secreting lamina propria plasma cells are tuned by local stimuli is completely unknown. Although antibody secretion is considered to be imprinted during B cell differentiation and therefore largely unaffected by environmental changes, a rapid modulation of IgA levels in response to intestinal fluctuations might be beneficial to the host. In the present study, we showed that dietary cholesterol absorption and commensal recognition by duodenal intestinal epithelial cells lead to the production of oxysterols, evolutionarily conserved lipids with immunomodulatory functions. Using conditional cholesterol 25-hydroxylase deleter mouse line we demonstrated that 7α,25-dihydroxycholesterol from epithelial cells is critical to restrain IgA secretion against commensal- and pathogen-derived antigens in the gut. Intestinal plasma cells sense oxysterols via the chemoattractant receptor GPR183 and couple their tissue positioning with IgA secretion. Our findings revealed a new mechanism linking dietary cholesterol and humoral immune responses centered around plasma cell localization for efficient mucosal protection.

Blocking AMPK ß1 myristoylation enhances AMPK activity and protects mice from high-fat diet-induced obesity and hepatic steatosis.

AMP-activated protein kinase (AMPK) is a master regulator of cellular energy homeostasis and a therapeutic target for metabolic diseases. Co/post-translational N-myristoylation of glycine-2 (Gly2) of the AMPK ß subunit has been suggested to regulate the distribution of the kinase between the cytosol and membranes through a "myristoyl switch" mechanism. However, the relevance of AMPK myristoylation for metabolic signaling in cells and in vivo is unclear. Here, we generated knockin mice with a Gly2-to-alanine point mutation of AMPKß1 (ß1-G2A). We demonstrate that non-myristoylated AMPKß1 has reduced stability but is associated with increased kinase activity and phosphorylation of the Thr172 activation site in the AMPK α subunit. Using proximity ligation assays, we show that loss of ß1 myristoylation impedes colocalization of the phosphatase PPM1A/B with AMPK in cells. Mice carrying the ß1-G2A mutation have improved metabolic health with reduced adiposity, hepatic lipid accumulation, and insulin resistance under conditions of high-fat diet-induced obesity.

STAT3 gain-of-function mutations connect leukemia with autoimmune disease by pathological NKG2Dhi CD8+ T cell dysregulation and accumulation.

The association between cancer and autoimmune disease is unexplained, exemplified by T cell large granular lymphocytic leukemia (T-LGL) where gain-of-function (GOF) somatic STAT3 mutations correlate with co-existing autoimmunity. To investigate whether these mutations are the cause or consequence of CD8+ T cell clonal expansions and autoimmunity, we analyzed patients and mice with germline STAT3 GOF mutations. STAT3 GOF mutations drove the accumulation of effector CD8+ T cell clones highly expressing NKG2D, the receptor for stress-induced MHC-class-I-related molecules. This subset also expressed genes for granzymes, perforin, interferon-γ, and Ccl5/Rantes and required NKG2D and the IL-15/IL-2 receptor IL2RB for maximal accumulation. Leukocyte-restricted STAT3 GOF was sufficient and CD8+ T cells were essential for lethal pathology in mice. These results demonstrate that STAT3 GOF mutations cause effector CD8+ T cell oligoclonal accumulation and that these rogue cells contribute to autoimmune pathology, supporting the hypothesis that somatic mutations in leukemia/lymphoma driver genes contribute to autoimmune disease.

Increased core body temperature exacerbates defective protein prenylation in mouse models of mevalonate kinase deficiency.

Mevalonate kinase deficiency (MKD) is characterized by recurrent fevers and flares of systemic inflammation, caused by biallelic loss-of-function mutations in MVK. The underlying disease mechanisms and triggers of inflammatory flares are poorly understood because of the lack of in vivo models. We describe genetically modified mice bearing the hypomorphic mutation p.Val377Ile (the commonest variant in patients with MKD) and amorphic, frameshift mutations in Mvk. Compound heterozygous mice recapitulated the characteristic biochemical phenotype of MKD, with increased plasma mevalonic acid and clear buildup of unprenylated GTPases in PBMCs, splenocytes, and bone marrow. The inflammatory response to LPS was enhanced in compound heterozygous mice and treatment with the NLRP3 inflammasome inhibitor MCC950 prevented the elevation of circulating IL-1ß, thus identifying a potential inflammasome target for future therapeutic approaches. Furthermore, lines of mice with a range of deficiencies in mevalonate kinase and abnormal prenylation mirrored the genotype-phenotype relationship in human MKD. Importantly, these mice allowed the determination of a threshold level of residual enzyme activity, below which protein prenylation is impaired. Elevated temperature dramatically but reversibly exacerbated the deficit in the mevalonate pathway and the defective prenylation in vitro and in vivo, highlighting increased body temperature as a likely trigger of inflammatory flares.

The retroelement Lx9 puts a brake on the immune response to virus infection.

The notion that mobile units of nucleic acid known as transposable elements can operate as genomic controlling elements was put forward over six decades ago1,2. However, it was not until the advancement of genomic sequencing technologies that the abundance and repertoire of transposable elements were revealed, and they are now known to constitute up to two-thirds of mammalian genomes3,4. The presence of DNA regulatory regions including promoters, enhancers and transcription-factor-binding sites within transposable elements5-8 has led to the hypothesis that transposable elements have been co-opted to regulate mammalian gene expression and cell phenotype8-14. Mammalian transposable elements include recent acquisitions and ancient transposable elements that have been maintained in the genome over evolutionary time. The presence of ancient conserved transposable elements correlates positively with the likelihood of a regulatory function, but functional validation remains an essential step to identify transposable element insertions that have a positive effect on fitness. Here we show that CRISPR-Cas9-mediated deletion of a transposable element-namely the LINE-1 retrotransposon Lx9c11-in mice results in an exaggerated and lethal immune response to virus infection. Lx9c11 is critical for the neogenesis of a non-coding RNA (Lx9c11-RegoS) that regulates genes of the Schlafen family, reduces the hyperinflammatory phenotype and rescues lethality in virus-infected Lx9c11-/- mice. These findings provide evidence that a transposable element can control the immune system to favour host survival during virus infection.

Mesoderm-derived PDGFRA+ cells regulate the emergence of hematopoietic stem cells in the dorsal aorta.

Mouse haematopoietic stem cells (HSCs) first emerge at embryonic day 10.5 (E10.5), on the ventral surface of the dorsal aorta, by endothelial-to-haematopoietic transition. We investigated whether mesenchymal stem cells, which provide an essential niche for long-term HSCs (LT-HSCs) in the bone marrow, reside in the aorta-gonad-mesonephros and contribute to the development of the dorsal aorta and endothelial-to-haematopoietic transition. Here we show that mesoderm-derived PDGFRA+ stromal cells (Mesp1der PSCs) contribute to the haemogenic endothelium of the dorsal aorta and populate the E10.5-E11.5 aorta-gonad-mesonephros but by E13.5 were replaced by neural-crest-derived PSCs (Wnt1der PSCs). Co-aggregating non-haemogenic endothelial cells with Mesp1der PSCs but not Wnt1der PSCs resulted in activation of a haematopoietic transcriptional programme in endothelial cells and generation of LT-HSCs. Dose-dependent inhibition of PDGFRA or BMP, WNT and NOTCH signalling interrupted this reprogramming event. Together, aorta-gonad-mesonephros Mesp1der PSCs could potentially be harnessed to manufacture LT-HSCs from endothelium.

IL-21 has a critical role in establishing germinal centers by amplifying early B cell proliferation.

The proliferation and differentiation of antigen-specific B cells, including the generation of germinal centers (GC), are prerequisites for long-lasting, antibody-mediated immune protection. Affinity for antigen determines B cell recruitment, proliferation, differentiation, and competitiveness in the response, largely through determining access to T cell help. However, how T cell-derived signals contribute to these outcomes is incompletely understood. Here, we report how the signature cytokine of follicular helper T cells, IL-21, acts as a key regulator of the initial B cell response by accelerating cell cycle progression and the rate of cycle entry, increasing their contribution to the ensuing GC. This effect occurs over a wide range of initial B cell receptor affinities and correlates with elevated AKT and S6 phosphorylation. Moreover, the resultant increased proliferation can explain the IL-21-mediated promotion of plasma cell differentiation. Collectively, our data establish that IL-21 acts from the outset of a T cell-dependent immune response to increase cell cycle progression and fuel cyclic re-entry of B cells, thereby regulating the initial GC size and early plasma cell output.

Interleukin-21, acting beyond the immunological synapse, independently controls T follicular helper and germinal center B cells.

Germinal centers (GCs), transient structures within B cell follicles and central to affinity maturation, require the coordinated behavior of T and B cells. IL-21, a pleiotropic T cell-derived cytokine, is key to GC biology through incompletely understood mechanisms. By genetically restricting production and receipt of IL-21 in vivo, we reveal how its independent actions on T and B cells combine to regulate the GC. IL-21 established the magnitude of the GC B cell response by promoting CD4+ T cell expansion and differentiation in a dose-dependent manner and with paracrine activity. Within GC, IL-21 specifically promoted B cell centroblast identity and, when bioavailability was high, plasma cell differentiation. Critically, these actions may occur irrespective of cognate T-B interactions, making IL-21 a general promoter of growth as distinct to a mediator of affinity-driven selection via synaptic delivery. This promiscuous activity of IL-21 explains the consequences of IL-21 deficiency on antibody-based immunity.

Immunizations with diverse sarbecovirus receptor-binding domains elicit SARS-CoV-2 neutralizing antibodies against a conserved site of vulnerability.

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second-generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor-binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that the neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies.

The unique biology of germinal center B cells.

Germinal center (GC) B cells are the source of the high-affinity, class-switched antibodies required for protective immunity. The unique biology of GC B cells involves iterative rounds of antibody gene somatic hypermutation coupled to multiple selection and differentiation pathways. Recent advances in areas such as single cell and gene editing technologies have shed new light upon these complex and dynamic processes. We review these findings here and integrate them into the current understanding of GC B cell replication and death, the retention of high-affinity and class-switched B cells in the GC, and differentiation into plasma and memory cell effectors. We also discuss how the biology of GC responses relates to vaccine effectiveness and outline current and future challenges in the field.

Osteocyte transcriptome mapping identifies a molecular landscape controlling skeletal homeostasis and susceptibility to skeletal disease.

Osteocytes are master regulators of the skeleton. We mapped the transcriptome of osteocytes from different skeletal sites, across age and sexes in mice to reveal genes and molecular programs that control this complex cellular-network. We define an osteocyte transcriptome signature of 1239 genes that distinguishes osteocytes from other cells. 77% have no previously known role in the skeleton and are enriched for genes regulating neuronal network formation, suggesting this programme is important in osteocyte communication. We evaluated 19 skeletal parameters in 733 knockout mouse lines and reveal 26 osteocyte transcriptome signature genes that control bone structure and function. We showed osteocyte transcriptome signature genes are enriched for human orthologs that cause monogenic skeletal disorders (P = 2.4 × 10-22) and are associated with the polygenic diseases osteoporosis (P = 1.8 × 10-13) and osteoarthritis (P = 1.6 × 10-7). Thus, we reveal the molecular landscape that regulates osteocyte network formation and function and establish the importance of osteocytes in human skeletal disease.

Potent SARS-CoV-2 binding and neutralization through maturation of iconic SARS-CoV-1 antibodies.

Antibodies against coronavirus spike protein potently protect against infection and disease, but whether such protection can be extended to variant coronaviruses is unclear. This is exemplified by a set of iconic and well-characterized monoclonal antibodies developed after the 2003 SARS outbreak, including mAbs m396, CR3022, CR3014 and 80R, which potently neutralize SARS-CoV-1, but not SARS-CoV-2. Here, we explore antibody engineering strategies to change and broaden their specificity, enabling nanomolar binding and potent neutralization of SARS-CoV-2. Intriguingly, while many of the matured clones maintained specificity of the parental antibody, new specificities were also observed, which was further confirmed by X-ray crystallography and cryo-electron microscopy, indicating that a limited set of VH antibody domains can give rise to variants targeting diverse epitopes, when paired with a diverse VL repertoire. Our findings open up over 15 years of antibody development efforts against SARS-CoV-1 to the SARS-CoV-2 field and outline general principles for the maturation of antibody specificity against emerging viruses.