Profiling the impact of anti-human CD20 monoclonal antibodies on lymphocyte B cell subsets and their precursors in the bone marrow and in lymphoid tissues in an immunocompromised mouse engrafted with human cells.

Ofatumumab (OFA) and ocrelizumab (OCRE) are two anti-CD20 monoclonal antibodies approved for the treatment of relapsing forms of multiple sclerosis due to their ability to deplete B lymphocytes. The aim of this study was to investigate the impact of these anti-hCD20 antibodies on B lymphocyte subsets in the circulation and in primary and secondary lymphoid organs in an immune system humanized mouse model (immunocompromised Rag2-/-Il2rg-/-CD47-/-) engrafted with human CD34+ hematopoietic stem cells. Three months after humanization, mice, which present adaptive immune cells only of human origin, were treated with OFA (0.3 mg/Kg; day 1, 3 and 5), or OCRE (10 mg/kg; day 1) or saline. Seven days after the last injection a robust (>90 %) decrease of circulating human CD20+ B lymphocytes was observed in both OFA- and OCRE-treated mice. A partial replenishment of B lymphocytes was detectable in blood 36 days from the last injection in OFA-treated mice, while no B lymphocytes could be detected in OCRE-treated mice up to 65 days post injection. Bone marrow profiling showed that during hCD20+ B cell depletion and replenishment, OCRE-treated mice preserved only preB-I cells in the bone marrow, while the bone marrow of OFA-treated mice presented both preB-I as well as preB-II cells, with the latter subset being the one closest to differentiate into immature B cells. These data together with changes in B cell distribution in other tissues suggest that ofatumumab preserve BM niches, critical for B lymphocyte replenishment, limiting potential side effects of the treatment associated with the increased risk of infection.

Lysosomes in the immunometabolic reprogramming of immune cells in atherosclerosis.

Lysosomes have a central role in the disposal of extracellular and intracellular cargo and also function as metabolic sensors and signalling platforms in the immunometabolic reprogramming of macrophages and other immune cells in atherosclerosis. Lysosomes can rapidly sense the presence of nutrients within immune cells, thereby switching from catabolism of extracellular material to the recycling of intracellular cargo. Such a fine-tuned degradative response supports the generation of metabolic building blocks through effectors such as mTORC1 or TFEB. By coupling nutrients to downstream signalling and metabolism, lysosomes serve as a crucial hub for cellular function in innate and adaptive immune cells. Lysosomal dysfunction is now recognized to be a hallmark of atherogenesis. Perturbations in nutrient-sensing and signalling have profound effects on the capacity of immune cells to handle cholesterol, perform phagocytosis and efferocytosis, and limit the activation of the inflammasome and other inflammatory pathways. Strategies to improve lysosomal function hold promise as novel modulators of the immunoinflammatory response associated with atherosclerosis. In this Review, we describe the crosstalk between lysosomal biology and immune cell function and polarization, with a particular focus on cellular immunometabolic reprogramming in the context of atherosclerosis.

ASGR1 deficiency diverts lipids toward adipose tissue but results in liver damage during obesity.

BACKGROUND: Asialoglycoprotein receptor 1 (ASGR1), primarily expressed on hepatocytes, promotes the clearance and the degradation of glycoproteins, including lipoproteins, from the circulation. In humans, loss-of-function variants of ASGR1 are associated with a favorable metabolic profile and reduced incidence of cardiovascular diseases. The molecular mechanisms by which ASGR1 could affect the onset of metabolic syndrome and obesity are unclear. Therefore, here we investigated the contribution of ASGR1 in the development of metabolic syndrome and obesity. METHODS: ASGR1 deficient mice (ASGR1-/-) were subjected to a high-fat diet (45% Kcal from fat) for 20 weeks. The systemic metabolic profile, hepatic and visceral adipose tissue were characterized for metabolic and structural alterations, as well as for immune cells infiltration. RESULTS: ASGR1-/- mice present a hypertrophic adipose tissue with 41% increase in fat accumulation in visceral adipose tissue (VAT), alongside with alteration in lipid metabolic pathways. Intriguingly, ASGR1-/- mice exhibit a comparable response to an acute glucose and insulin challenge in circulation, coupled with notably decreased in circulating cholesterol levels. Although the liver of ASGR1-/- have similar lipid accumulation to the WT mice, they present elevated levels of liver inflammation and a decrease in mitochondrial function. CONCLUSION: ASGR1 deficiency impacts energetic homeostasis during obesity leading to improved plasma lipid levels but increased VAT lipid accumulation and liver damage.

Dendritic cell immunoreceptor 2 (DCIR2) deficiency decreases hepatic conventional dendritic cell content but not the progression of diet-induced obesity.

AIMS: Inflammatory pathways and immune system dysregulation participate in the onset and progression of cardiometabolic diseases. The dendritic cell immunoreceptor 2 (DCIR2) is a C-type lectin receptor mainly expressed by conventional type 2 dendritic cells, involved in antigen recognition and in the modulation of T cell response. Here, we investigated the effect of DCIR2 deficiency during the development of obesity. METHODS: DCIR2 KO mice and the WT counterpart were fed with high-fat diet (HFD) for 20 weeks. Weight gain, glucose and insulin tolerance were assessed, parallel to immune cell subset profiling and histological analysis. RESULTS: After HFD feeding, DCIR2 KO mice presented altered conventional dendritic cell distribution within the liver without affecting markers of hepatic inflammation. These observations were liver restricted, since immune profile of metabolic and lymphoid organs-namely adipose tissue, spleen and mesenteric lymph nodes-did not show differences between the two groups. This reflected in a similar metabolic profile of DCIR2 KO compared to WT mice, characterized by comparable body weight gain as well as adipose tissues, spleen, Peyer's patches and mesenteric lymph nodes weight at sacrifice. Also, insulin response was similar in both groups. CONCLUSION: Our data show that DCIR2 has a redundant role in the progression of diet-induced obesity and inflammation.

The glucose transporter 2 regulates CD8+ T cell function via environment sensing.

T cell activation is associated with a profound and rapid metabolic response to meet increased energy demands for cell division, differentiation and development of effector function. Glucose uptake and engagement of the glycolytic pathway are major checkpoints for this event. Here we show that the low-affinity, concentration-dependent glucose transporter 2 (Glut2) regulates the development of CD8+ T cell effector responses in mice by promoting glucose uptake, glycolysis and glucose storage. Expression of Glut2 is modulated by environmental factors including glucose and oxygen availability and extracellular acidification. Glut2 is highly expressed by circulating, recently primed T cells, allowing efficient glucose uptake and storage. In glucose-deprived inflammatory environments, Glut2 becomes downregulated, thus preventing passive loss of intracellular glucose. Mechanistically, Glut2 expression is regulated by a combination of molecular interactions involving hypoxia-inducible factor-1 alpha, galectin-9 and stomatin. Finally, we show that human T cells also rely on this glucose transporter, thus providing a potential target for therapeutic immunomodulation.

Gonadal sex vs genetic sex in experimental atherosclerosis.

Epidemiological data and interventional studies with hormone replacement therapy suggest that women, at least until menopause, are at decreased cardiovascular risk compared to men. Still the molecular mechanisms beyond this difference are debated and the investigation in experimental models of atherosclerosis has been pivotal to prove that the activation of the estrogen receptor is atheroprotective, despite not enough to explain the differences reported in cardiovascular disease between male and female. This casts also for investigating the importance of the sex chromosome complement (genetic sex) beyond the contribution of sex hormones (gonadal sex) on atherosclerosis. Aim of this review is to present the dualism between gonadal sex and genetic sex with a focus on the data available from experimental models. The molecular mechanisms driving changes in lipid metabolism, immuno-inflammatory reactivity and vascular response in males and females that affect atherosclerosis progression will be discussed.

Fibronectin extra domain a limits liver dysfunction and protects mice during acute inflammation.

Background and aim: The primary transcript of fibronectin (FN) undergoes alternative splicing to generate different isoforms, including FN containing the Extra Domain A (FN_EDA+), whose expression is regulated spatially and temporarily during developmental and disease conditions including acute inflammation. The role of FN_EDA+ during sepsis, however, remains elusive. Methods: Mice constitutively express the EDA domain of fibronectin (EDA+/+); lacking the FN EDA domain (EDA-/-) or with a conditional ablation of EDA + inclusion only in liver produced FN (alb-CRE+EDA floxed mice) thus expressing normal plasma FN were used. Systemic inflammation and sepsis were induced by either LPS injection (70 mg/kg) or by cecal ligation and puncture (CLP) Neutrophils isolated from septic patients were tested for neutrophil binding ability. Results: We observed that EDA+/+ were protected toward sepsis as compared to EDA-/- mice. Also alb-CRE+EDA floxed mice presented reduced survival, thus indicating a key role for EDA in protecting toward sepsis. This phenotype was associated with improved liver and spleen inflammatory profile. Ex vivo experiments showed that neutrophils bind to a larger extent to an FN_EDA + coated surface as compared to FN, thus potentially limiting their over-reactivity. Conclusions: Our study demonstrates that the inclusion of the EDA domain in fibronectin dampens the nflammatoryi consequences of sepsis.

The heterogeneous cellular landscape of atherosclerosis: Implications for future research and therapies. A collaborative review from the EAS young fellows.

Single cell technologies, lineage tracing mouse models and advanced imaging techniques unequivocally improved the resolution of the cellular landscape of atherosclerosis. Although the discovery of the heterogeneous nature of the cellular plaque architecture has undoubtedly improved our understanding of the specific cellular states in atherosclerosis progression, it also adds more complexity to current and future research and will change how we approach future drug development. In this review, we will discuss how the revolution of new single cell technologies allowed us to map the cellular networks in the plaque, but we will also address current (technological) limitations that confine us to identify the cellular drivers of the disease and to pinpoint a specific cell state, cell subset or cell surface antigen as new candidate drug target for atherosclerosis.

Dendritic cell marker Clec4a4 deficiency limits atherosclerosis progression.

Background and aims: Atherogenesis results from altered lipid metabolism and impaired immune response. Emerging evidence has suggested that dendritic cells (DCs) participate to atherosclerosis-related immune response, but their impact is scarcely characterized. Clec4a4 or DCIR2 (Dendritic cell immunoreceptor 2) is a C-type lectin receptor, mainly expressed by CD8α- DCs, able to modulate T cell immunity. However, whether this DC subset could play a role in the atherogenesis is still poorly understood. Thus, the aim of this study is to investigate whether the absence of Clec4a4 could affect atherosclerosis-related immune response and atherosclerosis itself. Methods: Dcir2 -/- Ldlr -/- and Ldlr -/- mice were fed a standard diet or cholesterol-enriched diet for 12 weeks. Subsequently, the profile of circulating and lymph nodes-resident immune cells was investigated together with the analysis of plasma lipid levels and atherosclerotic plaque extension in the aorta. Results: Here, we show that Clec4a4 expression is downregulated under hypercholesterolemia and its deficiency in Ldlr -/- mice results in the reduction of atherosclerotic plaque formation, together with altered lipid metabolism and impaired myeloid immune cell distribution. Conclusions: Our findings suggest a pro-atherosclerotic role of Clec4a4 in experimental atherosclerosis.

Neutral effect of SGLT2 inhibitors on lipoprotein metabolism: From clinical evidence to molecular mechanisms.

Sodium-glucose cotransporter-2 inhibitors (SGLT2i) are effective, well-tolerated, and safe glucose-lowering compounds for patients with type 2 diabetes mellitus (T2DM). SGLT2i benefit encompasses protection from heart and kidney failure, independently of the presence of diabetes. In addition, SGLT2i consistently reduce the risk of hospitalization for heart failure and, although with some heterogeneity between specific members of the class, favourably affect the risk of cardiovascular outcomes. The molecular mechanisms underlying the cardiovascular favourable effect are not fully clarified. Studies testing the efficacy of SGLT2i in human cohorts and experimental models of atherosclerotic cardiovascular disease (ASCVD) have reported significant differences in circulating levels and composition of lipoprotein classes. In randomized clinical trials, small but significant increases in low-density lipoprotein cholesterol (LDL-C) levels have been observed, with a still undefined clinical significance; on the other hand, favourable (although modest) effects on high-density lipoprotein cholesterol (HDL-C) and triglycerides have been reported. At the molecular level, glycosuria may promote a starving-like state that ultimately leads to a metabolic improvement through the mobilization of fatty acids from the adipose tissue and their oxidation for the production of ketone bodies. This, however, may also fuel hepatic cholesterol synthesis, thus inhibiting atherogenic lipoprotein uptake from the liver. Long-term studies collecting detailed information on lipid-lowering therapies at baseline and during the trials with SGLT2i, as well as regularly monitoring lipid profiles are warranted to disentangle the potential implications of SGLT2i in modulating lipoprotein-mediated atherosclerotic cardiovascular risk.

Mannose Receptor Deficiency Impacts Bone Marrow and Circulating Immune Cells during High Fat Diet Induced Obesity.

The mannose receptor C-type 1 (Mrc1) is a C-type lectin receptor expressed on the immune cells and sinusoidal endothelial cells (ECs) of several tissues, including the bone marrow (BM). Parallel to systemic metabolic alterations and hematopoietic cell proliferation, high-fat diet (HFD) feeding increases the expression of Mrc1 in sinusoidal ECs, thus calling for the investigation of its role in bone marrow cell reprogramming and the metabolic profile during obesity. Mrc1-/- mice and wild-type (WT) littermates were fed an HFD (45% Kcal/diet) for 20 weeks. Weight gain was monitored during the diet regimen and glucose and insulin tolerance were assessed. Extensive flow cytometry profiling, histological, and proteomic analyses were performed. After HFD feeding, Mrc1-/- mice presented impaired medullary hematopoiesis with reduced myeloid progenitors and mature cells in parallel with an increase in BM adipocytes compared to controls. Accordingly, circulating levels of neutrophils and pro-inflammatory monocytes decreased in Mrc1-/- mice together with reduced infiltration of macrophages in the visceral adipose tissue and the liver compared to controls. Liver histological profiling coupled with untargeted proteomic analysis revealed that Mrc1-/- mice presented decreased liver steatosis and the downregulation of proteins belonging to pathways involved in liver dysfunction. This profile was reflected by improved glucose and insulin response and reduced weight gain during HFD feeding in Mrc1-/- mice compared to controls. Our data show that during HFD feeding, mannose receptor deficiency impacts BM and circulating immune cell subsets, which is associated with reduced systemic inflammation and resistance to obesity development.

The low-density lipoprotein receptor-mTORC1 axis coordinates CD8+ T cell activation.

Activation of T cells relies on the availability of intracellular cholesterol for an effective response after stimulation. We investigated the contribution of cholesterol derived from extracellular uptake by the low-density lipoprotein (LDL) receptor in the immunometabolic response of T cells. By combining proteomics, gene expression profiling, and immunophenotyping, we described a unique role for cholesterol provided by the LDLR pathway in CD8+ T cell activation. mRNA and protein expression of LDLR was significantly increased in activated CD8+ compared to CD4+ WT T cells, and this resulted in a significant reduction of proliferation and cytokine production (IFNγ, Granzyme B, and Perforin) of CD8+ but not CD4+ T cells from Ldlr -/- mice after in vitro and in vivo stimulation. This effect was the consequence of altered cholesterol routing to the lysosome resulting in a lower mTORC1 activation. Similarly, CD8+ T cells from humans affected by familial hypercholesterolemia (FH) carrying a mutation on the LDLR gene showed reduced activation after an immune challenge.

Pancreatic PCSK9 controls the organization of the ß-cell secretory pathway via LDLR-cholesterol axis.

BACKGROUND: Cholesterol is central to pancreatic ß-cell physiology and alterations of its homeostasis contribute to ß-cell dysfunction and diabetes. Proper intracellular cholesterol levels are maintained by different mechanisms including uptake via the low-density lipoprotein receptor (LDLR). In the liver, the proprotein convertase subtilisin/kexin type 9 (PCSK9) routes the LDLR to lysosomes for degradation, thus limiting its recycling to the membrane. PCSK9 is also expressed in the pancreas and loss of function mutations of PCSK9 result in higher plasma glucose levels and increased risk of Type 2 diabetes mellitus. Aim of this study was to investigate whether PCSK9 also impacts ß-cells function. METHODS: Pancreas-specific Pcsk9 null mice (Pdx1Cre/Pcsk9 fl/fl) were generated and characterized for glucose tolerance, insulin release and islet morphology. Isolated Pcsk9-deficient islets and clonal ß-cells (INS1E) were employed to characterize the molecular mechanisms of PCSK9 action. RESULTS: Pdx1Cre/Pcsk9 fl/fl mice exhibited normal blood PCSK9 and cholesterol levels but were glucose intolerant and had defective insulin secretion in vivo. Analysis of PCSK9-deficient islets revealed comparable ß-cell mass and insulin content but impaired stimulated secretion. Increased proinsulin/insulin ratio, modifications of SNARE proteins expression and decreased stimulated­calcium dynamics were detected in PCSK9-deficient ß-cells. Mechanistically, pancreatic PCSK9 silencing impacts ß-cell LDLR expression and cholesterol content, both in vivo and in vitro. The key role of LDLR is confirmed by the demonstration that LDLR downregulation rescued the phenotype. CONCLUSIONS: These findings establish pancreatic PCSK9 as a novel critical regulator of the functional maturation of the ß-cell secretory pathway, via modulation of cholesterol homeostasis.

Crosstalk between dendritic cells and T lymphocytes during atherogenesis: Focus on antigen presentation and break of tolerance.

Atherosclerosis is a chronic disease resulting from an impaired lipid and immune homeostasis, where the interaction between innate and adaptive immune cells leads to the promotion of atherosclerosis-associated immune-inflammatory response. Emerging evidence has suggested that this response presents similarities to the reactivity of effector immune cells toward self-epitopes, often as a consequence of a break of tolerance. In this context, dendritic cells, a heterogeneous population of antigen presenting cells, play a key role in instructing effector T cells to react against foreign antigens and T regulatory cells to maintain tolerance against self-antigens and/or to patrol for self-reactive effector T cells. Alterations in this delicate balance appears to contribute to atherogenesis. The aim of this review is to discuss different DC subsets, and their role in atherosclerosis as well as in T cell polarization. Moreover, we will discuss how loss of T cell tolerogenic phenotype participates to the immune-inflammatory response associated to atherosclerosis and how a better understanding of these mechanisms might result in designing immunomodulatory therapies targeting DC-T cell crosstalk for the treatment of atherosclerosis-related inflammation.

Lack of ApoA-I in ApoEKO Mice Causes Skin Xanthomas, Worsening of Inflammation, and Increased Coronary Atherosclerosis in the Absence of Hyperlipidemia.

BACKGROUND: HDL (high-density lipoprotein) and its major protein component, apoA-I (apolipoprotein A-I), play a unique role in cholesterol homeostasis and immunity. ApoA-I deficiency in hyperlipidemic, atheroprone mice was shown to drive cholesterol accumulation and inflammatory cell activation/proliferation. The present study was aimed at investigating the impact of apoA-I deficiency on lipid deposition and local/systemic inflammation in normolipidemic conditions. METHODS: ApoE deficient mice, apoE/apoA-I double deficient (DKO) mice, DKO mice overexpressing human apoA-I, and C57Bl/6J control mice were fed normal laboratory diet until 30 weeks of age. Plasma lipids were quantified, atherosclerosis development at the aortic sinus and coronary arteries was measured, skin ultrastructure was evaluated by electron microscopy. Blood and lymphoid organs were characterized through histological, immunocytofluorimetric, and whole transcriptome analyses. RESULTS: DKO were characterized by almost complete HDL deficiency and by plasma total cholesterol levels comparable to control mice. Only DKO showed xanthoma formation and severe inflammation in the skin-draining lymph nodes, whose transcriptome analysis revealed a dramatic impairment in energy metabolism and fatty acid oxidation pathways. An increased presence of CD4+ T effector memory cells was detected in blood, spleen, and skin-draining lymph nodes of DKO. A worsening of atherosclerosis at the aortic sinus and coronary arteries was also observed in DKO versus apoE deficient. Human apoA-I overexpression in the DKO background was able to rescue the skin phenotype and halt atherosclerosis development. CONCLUSIONS: HDL deficiency, in the absence of hyperlipidemia, is associated with severe alterations of skin morphology, aortic and coronary atherosclerosis, local and systemic inflammation.

Loss of voltage-gated hydrogen channel 1 expression reveals heterogeneous metabolic adaptation to intracellular acidification by T cells.

Voltage-gated hydrogen channel 1 (Hvcn1) is a voltage-gated proton channel, which reduces cytosol acidification and facilitates the production of ROS. The increased expression of this channel in some cancers has led to proposing Hvcn1 antagonists as potential therapeutics. While its role in most leukocytes has been studied in depth, the function of Hvcn1 in T cells remains poorly defined. We show that Hvcn1 plays a nonredundant role in protecting naive T cells from intracellular acidification during priming. Despite sharing overall functional impairment in vivo and in vitro, Hvcn1-deficient CD4+ and CD8+ T cells display profound differences during the transition from naive to primed T cells, including in the preservation of T cell receptor (TCR) signaling, cellular division, and death. These selective features result, at least in part, from a substantially different metabolic response to intracellular acidification associated with priming. While Hvcn1-deficient naive CD4+ T cells reprogram to rescue the glycolytic pathway, naive CD8+ T cells, which express high levels of this channel in the mitochondria, respond by metabolically compensating mitochondrial dysfunction, at least in part via AMPK activation. These observations imply heterogeneity between adaptation of naive CD4+ and CD8+ T cells to intracellular acidification during activation.

Genetically determined hypercholesterolaemia results into premature leucocyte telomere length shortening and reduced haematopoietic precursors.

AIMS: Leucocyte telomere length (LTL) shortening is a marker of cellular senescence and associates with increased risk of cardiovascular disease (CVD). A number of cardiovascular risk factors affect LTL, but the correlation between elevated LDL cholesterol (LDL-C) and shorter LTL is debated: in small cohorts including subjects with a clinical diagnosis of familial hypercholesterolaemia (FH). We assessed the relationship between LDL-C and LTL in subjects with genetic familial hypercholesterolaemia (HeFH) compared to those with clinically diagnosed, but not genetically confirmed FH (CD-FH), and normocholesterolaemic subjects. METHODS AND RESULTS: LTL was measured in mononuclear cells-derived genomic DNA from 206 hypercholesterolaemic subjects (135 HeFH and 71 CD-FH) and 272 controls. HeFH presented shorter LTL vs. controls (1.27 ± 0.07 vs. 1.59 ± 0.04, P = 0.045). In particular, we found shorter LTL in young HeFH as compared to young controls (<35 y) (1.34 ± 0.08 vs. 1.64 ± 0.08, P = 0.019); moreover, LTL was shorter in statin-naïve HeFH subjects as compared to controls (1.23 ± 0.08 vs. 1.58 ± 0.04, P = 0.001). HeFH subjects presented shorter LTL compared to LDL-C matched CD-FH (1.33 ± 0.05 vs. 1.55 ± 0.08, P = 0.029). Shorter LTL was confirmed in leucocytes of LDLR-KO vs. wild-type mice and associated with lower abundance of long-term haematopoietic stem and progenitor cells (LT-HSPCs) in the bone marrow. Accordingly, HeFH subjects presented lower circulating haematopoietic precursors (CD34 + CD45dim cells) vs. CD-FH and controls. CONCLUSIONS: We found (i) shorter LTL in genetically determined hypercholesterolaemia, (ii) lower circulating haematopoietic precursors in HeFH subjects, and reduced bone marrow resident LT-HSPCs in LDLR-KO mice. We support early cellular senescence and haematopoietic alterations in subjects with FH.