Innate immune cell-intrinsic ketogenesis is dispensable for organismal metabolism and age-related inflammation.

Aging is accompanied by chronic low-grade inflammation, but the mechanisms that allow this to persist are not well understood. Ketone bodies are alternative fuels produced when glucose is limited and improve indicators of healthspan in aging mouse models. Moreover, the most abundant ketone body, ß-hydroxybutyrate, inhibits the NLRP3 inflammasome in myeloid cells, a key potentiator of age-related inflammation. Given that myeloid cells express ketogenic machinery, we hypothesized this pathway may serve as a metabolic checkpoint of inflammation. To test this hypothesis, we conditionally ablated ketogenesis by disrupting expression of the terminal enzyme required for ketogenesis, 3-Hydroxy-3-Methylglutaryl-CoA Lyase (HMGCL). By deleting HMGCL in the liver, we validated the functional targeting and establish that the liver is the only organ that can produce the life-sustaining quantities of ketone bodies required for survival during fasting or ketogenic diet feeding. Conditional ablation of HMGCL in neutrophils and macrophages had modest effects on body weight and glucose tolerance in aging but worsened glucose homeostasis in myeloid cell-specific Hmgcl-deficient mice fed a high-fat diet. Our results suggest that during aging, liver-derived circulating ketone bodies might be more important for deactivating the NLRP3 inflammasome and controlling organismal metabolism.

Actin dynamics regulation by TTC7A/PI4KIIIα limits DNA damage and cell death under confinement.

BACKGROUND: The actin cytoskeleton has a crucial role in the maintenance of the immune homeostasis by controlling various cellular processes, including cell migration. Mutations in TTC7A have been described as the cause of a primary immunodeficiency associated to different degrees of gut involvement and alterations in the actin cytoskeleton dynamics. OBJECTIVES: This study investigates the impact of TTC7A deficiency in immune homeostasis. In particular, the role of the TTC7A/phosphatidylinositol 4 kinase type III α pathway in the control of leukocyte migration and actin dynamics. METHODS: Microfabricated devices were leveraged to study cell migration and actin dynamics of murine and patient-derived leukocytes under confinement at the single-cell level. RESULTS: We show that TTC7A-deficient lymphocytes exhibit an altered cell migration and reduced capacity to deform through narrow gaps. Mechanistically, TTC7A-deficient phenotype resulted from impaired phosphoinositide signaling, leading to the downregulation of the phosphoinositide 3-kinase/AKT/RHOA regulatory axis and imbalanced actin cytoskeleton dynamics. TTC7A-associated phenotype resulted in impaired cell motility, accumulation of DNA damage, and increased cell death in dense 3-dimensional gels in the presence of chemokines. CONCLUSIONS: These results highlight a novel role of TTC7A as a critical regulator of lymphocyte migration. Impairment of this cellular function is likely to contribute to the pathophysiology underlying progressive immunodeficiency in patients.

Ttc7a regulates hematopoietic stem cell functions while controlling the stress-induced response.

The molecular machinery that regulates the balance between self-renewal and differentiation properties of hematopoietic stem cells (HSC) has yet to be characterized in detail. Here we found that the tetratricopeptide repeat domain 7 A (Ttc7a) protein, a putative scaffold protein expressed by HSC, acts as an intrinsic regulator of the proliferative response and the self-renewal potential of murine HSC in vivo Loss of Ttc7a consistently enhanced the competitive repopulating ability of HSC and their intrinsic capacity to replenish the hematopoietic system after serial cell transplantations, relative to wildtype cells. Ttc7a-deficient HSC exhibit a different transcriptomic profile for a set of genes controlling the cellular response to stress, which was associated with increased proliferation in response to chemically induced stress in vitro and myeloablative stress in vivo Our results therefore revealed a previously unrecognized role of Ttc7a as a critical regulator of HSC stemness. This role is related, at least in part, to regulation of the endoplasmic reticulum stress response.

A catalytic-independent role for the LUBAC in NF-κB activation upon antigen receptor engagement and in lymphoma cells.

Antigen receptor-mediated nuclear factor κB (NF-κB) activation relies on the formation of a large multi-protein complex that contains CARMA1, BCL10, and MALT1 (CBM complex). This signalosome is pirated in the activated B-cell-like subgroup of diffuse large B-cell lymphoma (ABC DLBCL) to drive aberrant NF-κB activation, thereby promoting cell survival and propagation. Using an unbiased proteomic approach, we screened for additional components of the CBM in lymphocytes. We found that the linear ubiquitin chain assembly complex (LUBAC), which was previously linked to cytokine-mediated NF-κB activation, dynamically integrates the CBM and marshals NF-κB optimal activation following antigen receptor ligation independently of its catalytic activity. The LUBAC also participates in preassembled CBM complex in cells derived from ABC DLBCL. Silencing the LUBAC reduced NF-κB activation and was toxic in ABC DLBCL cell lines. Thus, our findings reveal a role for the LUBAC during lymphocyte activation and in B-cell malignancy.

Nerve-associated macrophages control adipose homeostasis across lifespan and restrain age-related inflammation.

Age-related inflammation or inflammaging is a key mechanism that increases disease burden and may control lifespan. How adipose tissue macrophages (ATMs) control inflammaging is not well understood in part because the molecular identities of niche-specific ATMs are incompletely known. Using intravascular labeling to exclude circulating myeloid cells and subsequent single-cell sequencing with orthogonal validation, we define the diversity and alterations in niche resident ATMs through lifespan. Aging led to depletion of vessel-associated macrophages (VAMs), expansion of lipid-associated macrophages (LAMs), and emergence of a unique subset of CD38+ age-associated macrophages (AAMs) in visceral white adipose tissue (VAT). Interestingly, CD169+CD11c- ATMs are enriched in a subpopulation of nerve-associated macrophages (NAMs) that declines with age. Depletion of CD169+ NAMs in aged mice increases inflammaging and impairs lipolysis suggesting that they are necessary for preventing catecholamine resistance in VAT. These findings reveal specialized ATMs control adipose homeostasis and link inflammation to tissue dysfunction during aging.

Negative regulation of NF-κB signaling in T lymphocytes by the ubiquitin-specific protease USP34.

BACKGROUND: NF-κB is a master gene regulator involved in plethora of biological processes, including lymphocyte activation and proliferation. Reversible ubiquitinylation of key adaptors is required to convey the optimal activation of NF-κB. However the deubiquitinylases (DUBs), which catalyze the removal of these post-translational modifications and participate to reset the system to basal level following T-Cell receptor (TCR) engagement continue to be elucidated. FINDINGS: Here, we performed an unbiased siRNA library screen targeting the DUBs encoded by the human genome to uncover new regulators of TCR-mediated NF-κB activation. We present evidence that knockdown of Ubiquitin-Specific Protease 34 (USP34) selectively enhanced NF-κB activation driven by TCR engagement, similarly to siRNA against the well-characterized DUB cylindromatosis (CYLD). From a molecular standpoint, USP34 silencing spared upstream signaling but led to a more pronounced degradation of the NF-κB inhibitor IκBα, and culminated with an increased DNA binding activity of the transcription factor. CONCLUSIONS: Collectively, our data unveils USP34 as a new player involved in the fine-tuning of NF-κB upon TCR stimulation.

Chronic Intestinal Pseudo-Obstruction and Lymphoproliferative Syndrome as a Novel Phenotype Associated With Tetratricopeptide Repeat Domain 7A Deficiency.

Mutations in the tetratricopeptide repeat domain 7A (TTC7A) gene cause very early onset inflammatory bowel diseases (VOIBD) or multiple intestinal atresia associated with immune deficiency of various severities, ranging from combined immune deficiency to mild lymphopenia. In this manuscript, we report the clinical, biological and molecular features of a patient born from consanguineous parents, presenting with recurrent lymphoproliferative syndrome and pan-hypergammaglobulinemia associated with chronic intestinal pseudo obstruction (CIPO). Genetic screening revealed the novel c.974G>A (p.R325Q) mutation in homozygosity in the TTC7A gene. The patient's phenotype differs significantly from that previously associated with TTC7A deficiency in humans. It becomes closer to the one reported in the ttc7a-deficient mice that invariably develop a proliferative lymphoid and myeloid disorder. Functional studies showed that the extreme variability in the clinical phenotype couldn't be explained by the cellular phenotype. Indeed, the patient's TTC7A mutation, as well as the murine-ttc7 mutant, have the same functional impact on protein expression, DNA instability and chromatin compaction, as the other mutations that lead to classical TTC7A-associated phenotypes. Co-inheritance of genetic variants may also contribute to the unique nature of the patient's phenotype. The present case report shows that the clinical spectrum of TTC7A deficiency is much broader than previously suspected. Our findings should alert the physicians to consider screening of TTC7A mutations in patients with lymphoproliferative syndrome and hypergammaglobulinemia and/or chronic intestinal pseudo-obstruction.

Tetratricopeptide repeat domain 7A is a nuclear factor that modulates transcription and chromatin structure.

A loss-of-function mutation in tetratricopeptide repeat domain 7A (TTC7A) is a recently identified cause of human intestinal and immune disorders. However, clues to related underlying molecular dysfunctions remain elusive. It is now shown based on the study of TTC7A-deficient and wild-type cells that TTC7A is an essential nuclear protein. It binds to chromatin, preferentially at actively transcribed regions. Its depletion results in broad range of epigenomic changes at proximal and distal transcriptional regulatory elements and in altered control of the transcriptional program. Loss of WT_TTC7A induces general decrease in chromatin compaction, unbalanced cellular distribution of histones, higher nucleosome accessibility to nuclease digestion along with genome instability, and reduced cell viability. Our observations characterize for the first time unreported functions for TTC7A in the nucleus that exert a critical role in chromatin organization and gene regulation to safeguard healthy immune and intestinal status.