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Several independent lines of evidence suggest that megakaryocytes are dysfunctional in severe COVID-19. Herein, we characterized peripheral circulating megakaryocytes in a large cohort of COVID-19 inpatients and correlated subpopulation frequencies with clinical outcomes. Using peripheral blood, we show that megakaryocytes are increased in the systemic circulation in COVID-19, and we identify and validate S100A8/A9 as a defining marker of megakaryocyte dysfunction. We further reveal a subpopulation of S100A8/A9+ megakaryocytes that contain SARS-CoV-2 protein and RNA. Using flow cytometry of peripheral blood and in vitro studies on SARS-CoV-2 infected primary human megakaryocytes, we demonstrate that megakaryocytes can transfer viral antigens to emerging platelets. Mechanistically, we show that SARS-CoV-2 containing megakaryocytes are NFκB-activated, via p65 and p52, express NFκB-mediated cytokines, IL-6 and IL-1β, and display high surface expression of TLR2 and TLR4, canonical drivers of NFκB. In a cohort of 218 COVID-19 inpatients, we correlate frequencies of megakaryocyte subpopulations with clinical outcomes and show that SARS-CoV-2 containing megakaryocytes are a strong risk factor for mortality and multi-organ injury, including respiratory failure, mechanical ventilation, acute kidney injury, thrombotic events, and ICU admission. Further, we show that SARS-CoV-2+ megakaryocytes are present in lung and brain autopsy tissues from deceased COVID-19 donors. This study offers the first evidence implicating SARS-CoV-2+ peripheral megakaryocytes in severe disease and suggests that circulating megakaryocytes warrant investigation in inflammatory disorders beyond COVID-19.

Human induced pluripotent stem cells (hiPSCs) were differentiated into a specific mesoderm subset characterized by KDR + CD56 + APLNR + (KNA + ) expression. KNA + cells had high clonal proliferative potential and specification into endothelial colony-forming cell (ECFCs) phenotype. KNA + cells differentiated into perfused blood vessels when implanted subcutaneously into the flank of nonobese diabetic/severe combined immunodeficient mice and when injected into the vitreous of type 2 diabetic mice ( db/db mice). Transcriptomic analysis showed that differentiation of hiPSCs derived from diabetics into KNA + cells was sufficient to change baseline differences in gene expression caused by the diabetic status and reprogram diabetic cells to a pattern similar to KNA + cells derived from nondiabetic hiPSCs. Proteomic array studies performed on retinas of db/db mice injected with either control or diabetic donor–derived KNA + cells showed correction of aberrant signaling in db/db retinas toward normal healthy retina. These data provide “proof of principle” that KNA + cells restore perfusion and correct vascular dysfunction in db/db mice.

Discovery of a biomarker for patients at high risk of progression to severe Coronavirus Disease 2019 (COVID-19) is critical for clinical management, particularly in areas of the world where widespread vaccine distribution and herd immunity have yet to be achieved. Herein, we characterize peripheral blood from 218 COVID-19 patients with flow cytometry and provide evidence that megakaryocytes are a target for infection by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). We demonstrate a positive correlation between infected megakaryocytes expressing the protein calprotectin (also called S100A8/A9), a known marker of COVID-19 severity. Additionally, we show that infected, calprotectin expressing megakaryocytes are correlated with COVID-19 severity and are a prognostic indicator of 30-day clinical outcomes including respiratory failure, thrombotic events, acute kidney injury (AKI), ICU admission, and mechanical ventilation. These findings represent a novel SARS-CoV-2 infection target with significant clinical implications as a biomarker for clinical outcomes associated with severe COVID-19.

The gut is a well-established route of infection and target for viral damage by SARS-CoV-2. This is supported by the clinical observation that about half of COVID-19 patients exhibit gastrointestinal (GI) symptoms. We asked whether the analysis of plasma could provide insight into gut barrier dysfunction in patients with COVID-19 infection. Plasma samples of COVID-19 patients (n=30) and healthy control (n=16) were collected during hospitalization. Plasma microbiome was analyzed using 16S rRNA sequencing, metatranscriptomic analysis, and gut permeability markers including FABP-2, PGN and LPS in both patient cohorts. Almost 65% (9 out 14) COVID-19 patients showed abnormal presence of gut microbes in their bloodstream. Plasma samples contained predominately Proteobacteria, Firmicutes, and Actinobacteria. The abundance of gram-negative bacteria (Acinetobacter, Nitrospirillum, Cupriavidus, Pseudomonas, Aquabacterium, Burkholderia, Caballeronia, Parabhurkholderia, Bravibacterium, and Sphingomonas) was higher than the gram-positive bacteria (Staphylococcus and Lactobacillus) in COVID-19 subjects. The levels of plasma gut permeability markers FABP2 (1282±199.6 vs 838.1±91.33; p=0.0757), PGN (34.64±3.178 vs 17.53±2.12; pvs 249.6±17.06; p=0.0049) were higher in COVID-19 patients compared to healthy subjects. These findings support that the intestine may represent a source for bacteremia and may contribute to worsening COVID-19 outcomes. Therapies targeting the gut and prevention of gut barrier defects may represent a strategy to improve outcomes in COVID-19 patients.

AIMS/HYPOTHESIS: Hypothalamic inflammation and sympathetic nervous system hyperactivity are hallmark features of the metabolic syndrome and type 2 diabetes. Hypothalamic inflammation may aggravate metabolic and immunological pathologies due to extensive sympathetic activation of peripheral tissues. Loss of somatostatinergic (SST) neurons may contribute to enhanced hypothalamic inflammation. METHODS: The present data show that leptin receptor-deficient (db/db) mice exhibit reduced hypothalamic SST neurons, particularly in the periventricular nucleus. We model this finding, using adeno-associated virus delivery of diphtheria toxin subunit A (DTA) driven by an SST-cre system to deplete these neurons in Sst(cre/gfp) mice (SST-DTA). RESULTS: SST-DTA mice exhibit enhanced hypothalamic c-Fos expression and brain inflammation as demonstrated by microglial and astrocytic activation. Bone marrow from SST-DTA mice undergoes skewed haematopoiesis, generating excess granulocyte-monocyte progenitors and increased proinflammatory (C-C chemokine receptor type 2; CCR2(hi)) monocytes. SST-DTA mice exhibited a ‘diabetic retinopathy-like’ phenotype: reduced visual function by optokinetic response (0.4 vs 0.25 cycles/degree; SST-DTA vs control mice); delayed electroretinogram oscillatory potentials; and increased percentages of retinal monocytes. Finally, mesenteric visceral adipose tissue from SST-DTA mice was resistant to catecholamine-induced lipolysis, displaying 50% reduction in isoprenaline (isoproterenol)-induced lipolysis compared with control littermates. Importantly, hyperglycaemia was not observed in SST-DTA mice. CONCLUSIONS/INTERPRETATION: The isolated reduction in hypothalamic SST neurons was able to recapitulate several hallmark features of type 2 diabetes in disease-relevant tissues.

Hypothalamic inflammation and sympathetic nervous system hyperactivity are hallmark features of metabolic syndrome and type 2 diabetes. Hypothalamic inflammation may aggravate metabolic and immunologic pathologies due to extensive sympathetic activation of peripheral tissues. Loss of somatostatinergic (SST) neurons may contribute to enhanced hypothalamic inflammation. The present data show that leptin receptor deficient (db/db) mice exhibit reduced hypothalamic somatostatinergic cells, particularly in the periventricular nucleus. We model this finding, using adeno-associated virus (AAV) delivery of diphtheria toxin (DTA) driven by an SST-cre system to deplete these cells in SSTcre/gfp mice (SST-DTA). SST-DTA mice exhibit enhanced hypothalamic c-fos expression and brain inflammation as demonstrated by microglial and astrocytic activation. Bone marrow from SST-DTA mice undergoes skewed hematopoiesis, generating excess granulocyte-monocyte precursors and increased pro-inflammatory (CCR2hi) monocytes. Visceral adipose tissue from DTA-treated animals was resistant to catecholamine induced lipolysis. Finally, SST-DTA mice exhibited a “diabetic retinopathy like” phenotype: reduced visual function by optokinetic response and electroretinogram, as well as increased percentages of retinal monocytes. Importantly, hyperglycemia was not observed in SST-DTA mice. Thus, the isolated reduction in hypothalamic somatostatinergic neurons was able to recapitulate several hallmark features of type 2 diabetes in disease relevant tissues.

AIMS/HYPOTHESIS: Homo Sapiens evolved under conditions of intermittent food availability and prolonged fasting between meals. Periods of fasting are important for recovery from meal-induced oxidative and metabolic stress, and tissue repair. Constant high energy-density food availability in present-day society contributes to the pathogenesis of chronic diseases, including diabetes and its complications, with intermittent fasting (IF) and energy restriction shown to improve metabolic health. We have previously demonstrated that IF prevents the development of diabetic retinopathy in a mouse model of type 2 diabetes (db/db); however the mechanisms of fasting-induced health benefits and fasting-induced risks for individuals with diabetes remain largely unknown. Sirtuin 1 (SIRT1), a nutrient-sensing deacetylase, is downregulated in diabetes. In this study, the effect of SIRT1 stimulation by IF, fasting mimicking cell culture conditions (FMC) or pharmacological treatment using SRT1720 was evaluated on systemic and retinal metabolism, systemic and retinal inflammation and vascular and bone marrow damage. METHODS: The effects of IF were modelled in vivo using db/db mice and in vitro using bovine retinal endothelial cells or rat retinal neuroglial/precursor R28 cell line serum starved for 24 h. mRNA expression was analysed by quantitative PCR (qPCR). SIRT1 activity was measured via histone deacetylase activity assay. NR1H3 (also known as Liver X receptor alpha (LXRα)) acetylation was measured via western blot analysis. RESULTS: IF increased Sirt1 mRNA expression in mouse liver and retina when compared with non-fasted animals. IF also increased SIRT1 activity eightfold in mouse retina while FMC increased SIRT1 activity and expression in retinal endothelial cells when compared with control. Sirt1 expression was also increased twofold in neuronal retina progenitor cells (R28) after FMC treatment. Moreover, FMC led to SIRT1-mediated LXRα deacetylation and subsequent 2.4-fold increase in activity, as measured by increased mRNA expression of the genes encoding ATP-binding cassette transporter (Abca1 and Abcg1). These changes were reduced when retinal endothelial cells expressing a constitutively acetylated LXRα mutant were tested. Increased SIRT1/LXR/ABC-mediated cholesterol export resulted in decreased retinal endothelial cell cholesterol levels. Direct activation of SIRT1 by SRT1720 in db/db mice led to a twofold reduction of diabetes-induced inflammation in the retina and improved diabetes-induced visual function impairment, as measured by electroretinogram and optokinetic response. In the bone marrow, there was prevention of diabetes-induced myeloidosis and decreased inflammatory cytokine expression. CONCLUSIONS/INTERPRETATION: Taken together, activation of SIRT1 signalling by IF or through pharmacological activation represents an effective therapeutic strategy that provides a mechanistic link between the advantageous effects associated with fasting regimens and prevention of microvascular and bone marrow dysfunction in diabetes.

Diabetic dyslipidemia, common in type 1 and type 2 diabetes (T2D), contributes to the pathogenesis of DR. LXR activation enhances cholesterol removal by reverse cholesterol transport (RCT) and reduces inflammatory gene expression. Systemic inflammation in diabetes is mediated, in part, by myeloidosis in the bone marrow (BM), which contributes to the pathogenesis of DR. We sought to test whether the progression of DR could be reduced by administering the novel LXR modulator, DMHCA (8mg/kg/body weight/day), in a T2D mouse model for 6 months. Retinal tissue was assessed by flow cytometry and Mass spectroscopy. LXR activation by DMHCA reduced cholesterol levels through the conversion to oxysterols and demosterol and was associated with a reduction of inflammatory cells in the retina and systemic circulation. Increased levels of long-term (17±1 vs. 11±2 db/db) and decrease levels of multipotent progenitors (MPP) (35±1 vs. 41±2 db/db) were noted in total bone marrow cells from db/db mice. DMHCA reduced the production of granulocyte-progenitors (GMP) (16 ± 1 vs. 22±3 db/db) and restored levels of NRF-2 (1±0.2 vs. 3±1 db/db) in bone marrow (BM) of db/db. DMHCA increased the number of myeloid angiogenic cells in the systemic circulation of db/db (0.04%±0.003 vs. 0.01%±0.002 db/db). Single cell RNA-seq analysis was performed using hematopoietic stem cells (HSCs) from untreated db/db and DMHCA treated db/db bone marrow. DMHCA treated HSCs showed upregulated genes associated with the activator protein 1 (AP-1) complex. AP-1 complexes regulate different stages of HSC differentiation along most of the myeloid lineage. Together, these findings reveal that LXR activation, via DMHCA treatment, decreases systemic and tissue-specific inflammation by controlling the production of pro-inflammatory stem cells and reducing oxidative injury in the BM of T2D mice. Disclosure C.P. Vieira: None. S.D. Fortmann: None. A. Longhini: None. T.A. Lydic: None. J.V. Busik: None. M.B. Grant: None. Funding National Institutes of Health (EY025383, EY012601)

In diabetic dyslipidemia, cholesterol accumulates in the plasma membrane, decreasing fluidity and thereby suppressing the ability of cells to transduce ligand-activated signaling pathways. Liver X receptors (LXRs) are the main cellular mechanism by which intracellular cholesterol is regulated and play important roles in inflammation and disease pathogenesis. N,N-dimethyl-3β-hydroxy-cholenamide (DMHCA), a selective LXR agonist, specifically activates the cholesterol efflux arm of the LXR pathway without stimulating triglyceride synthesis. Thus, DMHCA possesses superior clinical potential as a cholesterol lowering agent than current LXR pan-agonist. In this study, we use a multi-systems approach to understand the effects and molecular mechanisms of DMHCA treatment in type 2 diabetic db/db mice and human -derived circulating angiogenic cells (CACs), which are vascular reparative cells. We find that DMHCA is sufficient to correct the retina-bone marrow (BM) axis in diabetes, thereby restoring retinal structure, function, and cholesterol homeostasis, rejuvenating membrane fluidity in circulating vascular reparative cells, hampering systemic inflammation, and correcting BM dysfunction. Using single-cell RNA-seq on lineage-sca1+cKit+(LSK) hematopoietic stem cells (HSCs) from untreated and DMHCA-treated diabetic mice, we provide novel insights into hematopoiesis and reveal DMHCA’s mechanism of action in correcting diabetic HSCs by reducing myeloidosis and increasing CACs and erythrocyte progenitors. Taken together, these findings demonstrate the broad and pleiotropic effects of DMHCA treatment, which has exciting potential to correct the retina-BM axis in diabetic subjects.

Human induced pluripotent stem cells (hiPSC) differentiated into a specific mesoderm subset expressing vascular endothelial growth factor receptor 2, neural cell adhesion molecule 1, and apelin G protein-coupled receptor APJ (called KNA+), possessed all of the phenotypic and functional endothelial colony forming cell potential that resides in differentiating hiPSC. Thus, we postulated KNA+ mesoderm treatment would correct diabetic retinal capillary vasodegeneration. KNA+ cells derived from diabetic (D) and non-diabetic (N) patient-derived hiPSC displayed comparable phenotypic and functional properties. N-KNA+ or D-KNA+ intravitreal injections into type II diabetic ( db/db ) murine eyes led to retinal vascular engraftment, showed no toxicity, visual acuity was significantly improved, and diabetes-induced scotopic and photopic ERG defects were corrected. Proteomic arrays revealed that hiPSC-derived KNA+ cell administration restored several aberrant cell signaling pathways in the neural retina. These results support the efficacy of both hiPSC-derived D-KNA+ and N-KNA+ cells in correcting vasodegeneration in the diabetic murine retina.

SummaryIn diabetes, the retina, a tissue with unique metabolic needs, demonstrates dysregulation of the intricate balance between nutrient availability and utilization. This results in cholesterol accumulation, pro-inflammatory and pro-apoptotic changes, and consequently neurovascular damage. Sirtuin 1 (SIRT1), a nutrient sensing deacetylase, is downregulated in the diabetic retina. In this study, the effect of SIRT1 stimulation by fasting or by pharmacological activation using SRT1720, was evaluated on retinal cholesterol metabolism, inflammation and neurovascular damage. SIRT1 activation, in retinal endothelial cells (REC) and neuronal retinal progenitor cells (R28), led to Liver X Receptor alpha (LXRα) deacetylation and subsequent increased activity, as measured by increased ATP-binding cassette transporter (ABC) A1 and G1 mRNA expression. In turn, increased cholesterol export resulted in decreased REC cholesterol levels. SIRT1 activation also led to decreased inflammation. SIRT1 activation, in vivo, prevented diabetes-induced inflammation and vascular and neural degeneration. Diabetes-induced visual function impairment, as measured by electroretinogram and optokinetic response, was significantly improved as a result of SIRT1 activation. Taken together, activation of SIRT1 signaling is an effective therapeutic strategy that provides a mechanistic link between the advantageous effects associated with fasting regimes and prevention of diabetic retinopathy (DR).

SIRT1 is critical to vascular health and regulates glucose and lipid metabolism through its deacetylase activity. Type 2 diabetes-associated decreases in SIRT1 levels result in increased levels of acetylated LXR leading to reduced expression of reverse cholesterol transport genes and increased expression of NFκB-controlled pro-inflammatory genes. Diabetic circulating angiogenic cells (CAC) which are critical in vascular repair also demonstrate reduced SIRT1 and increasing its levels corrects CAC dysfunction (1). db/db mice were given SRT-1720 hydrochloride (100 mg/kg body weigh/day for 2 or 6 mo.) in their chow or fed normal chow beginning at diabetes onset. Retinal tissue, blood and bone marrow (BM) were collected. Electroretinograms (ERG) were performed at 2 and 6 mo. The BM supernatant was analyzed for cytokines. BM hematopoietic stem cells and CACs were assessed by flow cytometry. Retinal lipids were assessed by Mass Spectroscopy. At 2 mo, the retina of SRT1720-treated db/db mice demonstrated an increase in the ratio of total cholesterol/demosterol/lanosterol (197±85) compared to db/db (65±12. p Disclosure C.P. Vieira: None. A. Longhini: None. S. Li Calzi: None. M.D. DuPont: None. J.V. Busik: None. M.B. Grant: None. Funding National Institutes of Health (EY025383)

Numerous studies demonstrate the essential role of mesenchymal stem cells (MSCs) in the treatment of metabolic and inflammatory diseases, as these cells are known to modulate humoral and cellular immune responses. In this manuscript, we efficiently present two novel approaches to obtain MSCs from equine or human sources. In our first approach, we used electro-acupuncture as previously described by our group to mobilize MSCs into the peripheral blood of horses. For equine MSC collection, culture, and expansion, we used the Miltenyi Biotec CliniMACS Prodigy system of automated cell manufacturing. Using this system, we were able to generate appoximately 100 MSC colonies that exhibit surface marker expression of CD105 (92%), CD90 (85%), and CD73 (88%) within seven days of blood collection. Our second approach utilized the iPSC embryoid bodies from healthy or diabetic subjects where the iPSCs were cultured in standard media (endothelial + mesoderm basal media). After 21 days, the cells were FACS sorted and exhibited surface marker expression of CD105, CD90, and CD73. Both the equine cells and the human iPSC-derived MSCs were able to differentiate into adipogenic, osteogenic, and chondrogenic lineages. Both methods described simple and highly efficient methods to produce cells with surface markers phenotypically considered as MSCs and may, in the future, facilitate rapid production of MSCs with therapeutic potential.

Intermittent fasting (IF) protects against the development of metabolic diseases and cancer, but whether it can prevent diabetic microvascular complications is not known. In db/db mice, we examined the impact of long-term IF on diabetic retinopathy (DR). Despite no change in glycated hemoglobin, db/db mice on the IF regimen displayed significantly longer survival and a reduction in DR end points, including acellular capillaries and leukocyte infiltration. We hypothesized that IF-mediated changes in the gut microbiota would produce beneficial metabolites and prevent the development of DR. Microbiome analysis revealed increased levels of Firmicutes and decreased Bacteroidetes and Verrucomicrobia. Compared with db/db mice on ad libitum feeding, changes in the microbiome of the db/db mice on IF were associated with increases in gut mucin, goblet cell number, villi length, and reductions in plasma peptidoglycan. Consistent with the known modulatory effects of Firmicutes on bile acid (BA) metabolism, measurement of BAs demonstrated a significant increase of tauroursodeoxycholate (TUDCA), a neuroprotective BA, in db/db on IF but not in db/db on AL feeding. TGR5, the TUDCA receptor, was found in the retinal primary ganglion cells. Expression of TGR5 did not change with IF or diabetes. However, IF reduced retinal TNF-α mRNA, which is a downstream target of TGR5 activation. Pharmacological activation of TGR5 using INT-767 prevented DR in a second diabetic mouse model. These findings support the concept that IF prevents DR by restructuring the microbiota toward species producing TUDCA and subsequent retinal protection by TGR5 activation.

Previously, we showed that mesenchymal stem cells (MSC) can be mobilized into peripheral blood using electroacupuncture (EA) at acupoints, LI-4, LI-11, GV-14, and GV-20. The purpose of this study was to determine whether EA-mobilized MSC could be harvested and expanded in vitro to be used as an autologous cell therapy in horses. Peripheral blood mononuclear cells (PBMC) isolated from young and aged lame horses (n = 29) showed a marked enrichment for MSCs. MSC were expanded in vitro (n = 25) and administered intravenously at a dose of 50 x 106 (n = 24). Treatment resulted in significant improvement in lameness as assessed by the American Association of Equine Practitioners (AAEP) lameness scale (n = 23). MSCs exhibited immunomodulatory function by inhibition of lymphocyte proliferation and induction of IL-10. Intradermal testing showed no immediate or delayed immune reactions to MSC (1 x 106 to 1 x 104). In this study, we demonstrated an efficient, safe and reproducible method to mobilize and expand, in vitro, MSCs in sufficiently high concentrations for therapeutic administration. We confirm the immunomodulatory function of these cells in vitro. This non-pharmacological and non-surgical strategy for stem cell harvest has a broad range of biomedical applications and represents an improved clinically translatable and economical cell source for humans.