Optimization of the Irf8 +32-kb enhancer disrupts dendritic cell lineage segregation.

Autoactivation of lineage-determining transcription factors mediates bistable expression, generating distinct cell phenotypes essential for complex body plans. Classical type 1 dendritic cell (cDC1) and type 2 dendritic cell (cDC2) subsets provide nonredundant functions for defense against distinct immune challenges. Interferon regulatory factor 8 (IRF8), the cDC1 lineage-determining transcription factor, undergoes autoactivation in cDC1 progenitors to establish cDC1 identity, yet its expression is downregulated during cDC2 differentiation by an unknown mechanism. This study reveals that the Irf8 +32-kb enhancer, responsible for IRF8 autoactivation, is naturally suboptimized with low-affinity IRF8 binding sites. Introducing multiple high-affinity IRF8 sites into the Irf8 +32-kb enhancer causes a gain-of-function effect, leading to erroneous IRF8 autoactivation in specified cDC2 progenitors, redirecting them toward cDC1 and a novel hybrid DC subset with mixed-lineage phenotypes. Further, this also causes a loss-of-function effect, reducing Irf8 expression in cDC1s. These developmental alterations critically impair both cDC1-dependent and cDC2-dependent arms of immunity. Collectively, our findings underscore the significance of enhancer suboptimization in the developmental segregation of cDCs required for normal immune function.

Lipid kinase PIK3C3 maintains healthy brown and white adipose tissues to prevent metabolic diseases.

Adequate mass and function of adipose tissues (ATs) play essential roles in preventing metabolic perturbations. The pathological reduction of ATs in lipodystrophy leads to an array of metabolic diseases. Understanding the underlying mechanisms may benefit the development of effective therapies. Several cellular processes, including autophagy and vesicle trafficking, function collectively to maintain AT homeostasis. Here, we investigated the impact of adipocyte-specific deletion of the lipid kinase phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3) on AT homeostasis and systemic metabolism in mice. We report that PIK3C3 functions in all ATs and that its absence disturbs adipocyte autophagy and hinders adipocyte differentiation, survival, and function with differential effects on brown and white ATs. These abnormalities cause loss of white ATs, whitening followed by loss of brown ATs, and impaired "browning" of white ATs. Consequently, mice exhibit compromised thermogenic capacity and develop dyslipidemia, hepatic steatosis, insulin resistance, and type 2 diabetes. While these effects of PIK3C3 largely contrast previous findings with the autophagy-related (ATG) protein ATG7 in adipocytes, mice with a combined deficiency in both factors reveal a dominant role of the PIK3C3-deficient phenotype. We have also found that dietary lipid excess exacerbates AT pathologies caused by PIK3C3 deficiency. Surprisingly, glucose tolerance is spared in adipocyte-specific PIK3C3-deficient mice, a phenotype that is more evident during dietary lipid excess. These findings reveal a crucial yet complex role for PIK3C3 in ATs, with potential therapeutic implications.

Innate and Innate-like Effector Lymphocytes in Health and Disease.

Lymphocytes can be functionally partitioned into subsets belonging to the innate or adaptive arms of the immune system. Subsets of innate and innate-like lymphocytes may or may not express Ag-specific receptors of the adaptive immune system, yet they are poised to respond with innate-like speed to pathogenic insults but lack the capacity to develop classical immunological memory. These lymphocyte subsets display a number of common properties that permit them to integrate danger and stress signals dispatched by innate sensor cells to facilitate the generation of specialized effector immune responses tailored toward specific pathogens or other insults. In this review, we discuss the functions of distinct subsets of innate and innate-like lymphocytes. A better understanding of the mechanisms by which these cells are activated in different contexts, their interactions with other immune cells, and their role in health and disease may inform the development of new or improved immunotherapies.

IL-10-producing B cells are enriched in murine pericardial adipose tissues and ameliorate the outcome of acute myocardial infarction.

Acute myocardial infarction (MI) provokes an inflammatory response in the heart that removes damaged tissues to facilitate tissue repair/regeneration. However, overactive and prolonged inflammation compromises healing, which may be counteracted by antiinflammatory mechanisms. A key regulatory factor in an inflammatory response is the antiinflammatory cytokine IL-10, which can be produced by a number of immune cells, including subsets of B lymphocytes. Here, we investigated IL-10-producing B cells in pericardial adipose tissues (PATs) and their role in the healing process following acute MI in mice. We found that IL-10-producing B cells were enriched in PATs compared to other adipose depots throughout the body, with the majority of them bearing a surface phenotype consistent with CD5+ B-1a cells (CD5+ B cells). These cells were detected early in life, maintained a steady presence during adulthood, and resided in fat-associated lymphoid clusters. The cytokine IL-33 and the chemokine CXCL13 were preferentially expressed in PATs and contributed to the enrichment of IL-10-producing CD5+ B cells. Following acute MI, the pool of CD5+ B cells was expanded in PATs. These cells accumulated in the infarcted heart during the resolution of MI-induced inflammation. B cell-specific deletion of IL-10 worsened cardiac function, exacerbated myocardial injury, and delayed resolution of inflammation following acute MI. These results revealed enrichment of IL-10-producing B cells in PATs and a significant contribution of these cells to the antiinflammatory processes that terminate MI-induced inflammation. Together, these findings have identified IL-10-producing B cells as therapeutic targets to improve the outcome of MI.

PIK3C3/VPS34 keeps body fats healthy.

Adipose tissue, or body fat, plays a critical role in the maintenance of health and the development of metabolic diseases. The pathological expansion of adipose tissue during obesity and the pathological reduction of adipose tissue during lipodystrophy can lead to a similar array of metabolic diseases that include diabetes, but mechanisms remain to be fully defined. In our recent studies, we explored the contribution of the lipid kinase PIK3C3/VPS34 to adipose tissue health and metabolic disease. We found that adipocyte-specific PIK3C3/VPS34 deficiency causes defects in the differentiation, survival and functional properties of adipocytes, resulting in reduced adipose tissue mass, altered blood lipid levels, fatty liver disease, diabetes, and defective body temperature control. These abnormalities mirror those observed in patients with lipodystrophy. These findings identify adipocyte PIK3C3/VPS34 as a potential target for therapeutic intervention in metabolic diseases.

PIK3C3/VPS34 helps school T cells in the thymus.

The development of a broad repertoire of T cells in the immune system requires interaction of T cell receptors expressed by immature T cells with peptide/major histocompatibility complexes (MHCs) displayed by specialized epithelial cells in the thymus, in a process called T cell positive selection. Thymic epithelial cells (TECs) display unique antigen processing machinery which shapes the collection of self-peptides that drive positive selection. In our recent studies, we explored the contribution of the lipid kinase PIK3C3/VPS34 to the generation of positively selecting peptides in TECs. We found that TEC-specific PIK3C3/VPS34 facilitates the positive selection of CD4 but not CD8 T lineage cells, in a mechanism independent of its role in canonical macroautophagy/autophagy. Instead, we propose that PIK3C3/VPS34 alters vesicle trafficking in TECs that modulates lysosomal protease activity which, in turn, controls the generation of MHC class II-presented peptides optimized for positive selection of CD4 T cells.

Dendritic cell PIK3C3/VPS34 controls the pathogenicity of CNS autoimmunity independently of LC3-associated phagocytosis.

PIK3C3/VPS34 is a key player in macroautophagy/autophagy and MAP1LC3/LC3-associated phagocytosis (LAP), which play critical roles in dendritic cell (DC) function. In this study, we assessed the contribution of PIK3C3 to DC function during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). We found that Pik3c3-deficient DCs exhibit attenuated capacity to reactivate encephalitogenic T cells in the central nervous system, leading to reduced incidence and severity of EAE in DC-specific Pik3c3-deficient mice. Additionally, animals with a DC-specific deficiency in Rb1cc1/Fip200 but not Rubcn were protected against EAE, suggesting that the EAE phenotype of DC-specific Pik3c3-deficient mice is due to defective canonical autophagy rather than LAP. Collectively, our studies have revealed a critical role of PIK3C3 in DC function and the pathogenicity of these cells during EAE, with important implications for the development of immunotherapies for autoimmune diseases such as MS.Abbreviations: ATG: autophagy-related; CNS: central nervous system; DC: dendritic cell; DEG: differentially expressed gene; EAE: experimental autoimmune encephalomyelitis; LAP: LC3-associated phagocytosis; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MHC: major histocompatibility complex; MOG: myelin oligodendrocyte glycoprotein; MS: multiple sclerosis; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; ROS: reactive oxygen species.

Thymic epithelial cells require lipid kinase Vps34 for CD4 but not CD8 T cell selection.

The generation of a functional, self-tolerant T cell receptor (TCR) repertoire depends on interactions between developing thymocytes and antigen-presenting thymic epithelial cells (TECs). Cortical TECs (cTECs) rely on unique antigen-processing machinery to generate self-peptides specialized for T cell positive selection. In our current study, we focus on the lipid kinase Vps34, which has been implicated in autophagy and endocytic vesicle trafficking. We show that loss of Vps34 in TECs causes profound defects in the positive selection of the CD4 T cell lineage but not the CD8 T cell lineage. Utilizing TCR sequencing, we show that T cell selection in conditional mutants causes altered repertoire properties including reduced clonal sharing. cTECs from mutant mice display an increased abundance of invariant chain intermediates bound to surface MHC class II molecules, indicating altered antigen processing. Collectively, these studies identify lipid kinase Vps34 as an important contributor to the repertoire of selecting ligands processed and presented by TECs to developing CD4 T cells.

Autophagy-related protein PIK3C3/VPS34 controls T cell metabolism and function.

The PIK3C3/VPS34 subunit of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex is a key early player in macroautophagy/autophagy. In this study, we assessed the contribution of PIK3C3 to T cell metabolism and function. We found that Pik3c3-deficient T cells exhibited impaired cellular metabolism, and Pik3c3-deficient CD4+ T cells failed to differentiate into T helper 1 cells. These alterations were associated with reduced levels of active mitochondria upon T cell activation. In addition, conditional Pik3c3-deficient animals failed to mount autoreactive T cell responses and were resistant to experimental autoimmune encephalomyelitis (EAE). Interestingly, the deletion of Pik3c3 had little effect on the capacity of animals to clear tumor metastases. Collectively, our studies have revealed a critical role of PIK3C3 in T cell metabolism and the pathogenicity of these cells during EAE. Our findings also have important implications for the development of immunotherapies to treat multiple sclerosis and other inflammatory diseases by targeting PIK3C3.Abbreviations: CNS: central nervous system; DC: dendritic cell; DEG: differentially expressed gene; EAE: experimental autoimmune encephalomyelitis; ECAR: extracellular acidification rate; iNKT: invariant natural killer T; LAP: LC3-associated phagocytosis; LLC: Lewis lung carcinoma; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MDSC: myeloid-derived suppressor cell; MOG: myelin oligodendrocyte glycoprotein; NK: natural killer; OCR: oxygen consumption rate; PI: propidium iodide; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; RNA-seq: RNA-sequencing; TCR: T cell receptor; TMRE: tetramethylrhodamine ethyl ester perchlorate.

Pik3c3 deficiency in myeloid cells imparts partial resistance to experimental autoimmune encephalomyelitis associated with reduced IL-1ß production.

The PIK3C3/VPS34 subunit of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex plays a role in both canonical and noncanonical autophagy, key processes that control immune-cell responsiveness to a variety of stimuli. Our previous studies found that PIK3C3 is a critical regulator that controls the development, homeostasis, and function of dendritic and T cells. In this study, we investigated the role of PIK3C3 in myeloid cell biology using myeloid cell-specific Pik3c3-deficient mice. We found that Pik3c3-deficient macrophages express increased surface levels of major histocompatibility complex (MHC) class I and class II molecules. In addition, myeloid cell-specific Pik3c3 ablation in mice caused a partial impairment in the homeostatic maintenance of macrophages expressing the apoptotic cell uptake receptor TIM-4. Pik3c3 deficiency caused phenotypic changes in myeloid cells that were dependent on the early machinery (initiation/nucleation) of the classical autophagy pathway. Consequently, myeloid cell-specific Pik3c3-deficient animals showed significantly reduced severity of experimental autoimmune encephalomyelitis (EAE), a primarily CD4+ T-cell-mediated mouse model of multiple sclerosis (MS). This disease protection was associated with reduced accumulation of myelin-specific CD4+ T cells in the central nervous system and decreased myeloid cell IL-1ß production. Further, administration of SAR405, a selective PIK3C3 inhibitor, delayed disease progression. Collectively, our studies establish PIK3C3 as an important regulator of macrophage functions and myeloid cell-mediated regulation of EAE. Our findings also have important implications for the development of small-molecule inhibitors of PIK3C3 as therapeutic modulators of MS and other autoimmune diseases.

Role of autophagy in MHC class I-restricted antigen presentation.

Major histocompatibility complex (MHC) class I molecules present peptide antigens to MHC class I-restricted CD8+ T lymphocytes. The peptides loaded onto MHC class I molecules are typically derived from cytosolic antigens, which includes both self and foreign proteins. In addition to this classical MHC class I antigen presentation pathway, some cell types, especially dendritic cells can present antigens from exogenous sources to MHC class I-restricted CD8+ T cells, in a process called cross-presentation. A variety of cellular processes, including endocytosis, vesicle trafficking, and autophagy, play critical roles in these antigen presentation pathways. In this review article, we discuss the role of autophagy, an intracellular degradation system that delivers cytoplasmic constituents to lysosomes, in MHC class I-restricted antigen presentation. A mechanistic understanding of the role of autophagy-related proteins in MHC class I restricted antigen presentation may guide future efforts in manipulating autophagy to prevent or treat human disease.