Molecular pixelation: spatial proteomics of single cells by sequencing.

The spatial distribution of cell surface proteins governs vital processes of the immune system such as intercellular communication and mobility. However, fluorescence microscopy has limited scalability in the multiplexing and throughput needed to drive spatial proteomics discoveries at subcellular level. We present Molecular Pixelation (MPX), an optics-free, DNA sequence-based method for spatial proteomics of single cells using antibody-oligonucleotide conjugates (AOCs) and DNA-based, nanometer-sized molecular pixels. The relative locations of AOCs are inferred by sequentially associating them into local neighborhoods using the sequence-unique DNA pixels, forming >1,000 spatially connected zones per cell in 3D. For each single cell, DNA-sequencing reads are computationally arranged into spatial proteomics networks for 76 proteins. By studying immune cell dynamics using spatial statistics on graph representations of the data, we identify known and new patterns of spatial organization of proteins on chemokine-stimulated T cells, highlighting the potential of MPX in defining cell states by the spatial arrangement of proteins.

Antibodies against Phosphorylcholine and Malondialdehyde during the First Two Years of Life.

Abs against phosphorylcholine (anti-PC) and Abs against malondialdehyde (anti-MDA) may be protective in chronic inflammation, like atherosclerosis and cardiovascular disease. It is not known how they develop early in life. Ab titers were measured using ELISA in healthy women (n = 105; born into life study) and their children. Plasma samples were collected from the mothers before conception and from the children at birth as well as at 1 and 2 y after birth. Extracted Abs were compared using a proteomics de novo sequencing approach. It was observed that children were born with very low levels of IgM anti-PC, whereas IgM anti-MDA was present at birth. Both IgM anti-PC and anti-MDA increased during the first 2 y of life, but IgM anti-PC in contrast to IgM anti-MDA was still significantly lower than in the mothers. IgG anti-PC decreased after 1 y but reached similar levels as mothers' after 2 y, whereas IgG anti-MDA reached similar levels as mothers' already after 1 y. Proteomics peptide sequencing analysis indicated large peptide sequence variation without specific clone expression during the early stage of life compared with the adult stage for which specific peptide sequences dominated. IgM anti-PC levels develop much slower than anti-MDA and are still relatively low at 2 y. We hypothesize that anti-PC is developed by a combination of preprogramming and exposure to the external world, in which infectious agents may play a role. For anti-MDA, preprogramming is likely to play a major role and at an earlier stage than for anti-PC.

Insights From Liver-Humanized Mice on Cholesterol Lipoprotein Metabolism and LXR-Agonist Pharmacodynamics in Humans.

BACKGROUND AND AIMS: Genetically modified mice have been used extensively to study human disease. However, the data gained are not always translatable to humans because of major species differences. Liver-humanized mice (LHM) are considered a promising model to study human hepatic and systemic metabolism. Therefore, we aimed to further explore their lipoprotein metabolism and to characterize key hepatic species-related, physiological differences. APPROACH AND RESULTS: Fah-/- , Rag2-/- , and Il2rg-/- knockout mice on the nonobese diabetic (FRGN) background were repopulated with primary human hepatocytes from different donors. Cholesterol lipoprotein profiles of LHM showed a human-like pattern, characterized by a high ratio of low-density lipoprotein to high-density lipoprotein, and dependency on the human donor. This pattern was determined by a higher level of apolipoprotein B100 in circulation, as a result of lower hepatic mRNA editing and low-density lipoprotein receptor expression, and higher levels of circulating proprotein convertase subtilisin/kexin type 9. As a consequence, LHM lipoproteins bind to human aortic proteoglycans in a pattern similar to human lipoproteins. Unexpectedly, cholesteryl ester transfer protein was not required to determine the human-like cholesterol lipoprotein profile. Moreover, LHM treated with GW3965 mimicked the negative lipid outcomes of the first human trial of liver X receptor stimulation (i.e., a dramatic increase of cholesterol and triglycerides in circulation). Innovatively, LHM allowed the characterization of these effects at a molecular level. CONCLUSIONS: LHM represent an interesting translatable model of human hepatic and lipoprotein metabolism. Because several metabolic parameters displayed donor dependency, LHM may also be used in studies for personalized medicine.

IgM antibodies against malondialdehyde and phosphorylcholine in different systemic rheumatic diseases.

IgM antibodies against phosphorylcholine (anti-PC) and malondialdehyde (anti-MDA) may have protective properties in cardiovascular and rheumatic diseases. We here compare these antibodies in systemic rheumatic conditions and study their properties. Anti-PC and anti-MDA was measured using ELISA in patients with SLE (374), RA (354), Mixed connective tissue disease (MCTD, 77), Systemic sclerosis (SSc, 331), Sjögren's syndrome (SjS, 324), primary antiphospholipid syndrome (PAPs, 65), undifferentiated connective tissue disease (UCTD, 118) and 515 matched healthy controls (HC). Cardiovascular score (CV) was broadly defined based on clinical disease symptoms. Anti-PC and anti-MDA peptide/protein characterization were compared using a proteomics de novo sequencing approach. anti-MDA and anti-PC were extracted from total IgM. The proportion of Treg cells was determined by flow cytometry. The maximal difference between cases and controls was shown for MCTD: significantly lower IgM Anti-PC but not anti-MDA among patients (median 49.3RU/ml vs 70.4 in healthy controls, p(t-test) = 0.0037). IgM low levels were more prevalent in MCTD, SLE, SjS, SSc and UCTD. IgM anti-PC variable region profiles were different from and more homologous than anti-MDA. Anti-PC but not anti-MDA were significantly negatively correlated with CV in the whole patient group. In contrast to IgM anti-PC, anti-MDA did not promote polarization of Tregs. Taken together, Anti-PC is decreased in MCTD and also in SLE, SjS and SSc but not in other studied diseases. Anti-PC may thus differentiate between these. In contrast, anti-MDA did not show these differences between diseases studied. Anti-PC level is negatively correlated with CV in the patient group cohort. In contrast to anti-PC, anti-MDA did not promote Treg polarization. These findings could have both diagnostic and therapeutic implications, one possibility being active or passive immunization with PC in some rheumatic conditions.

Human IgM Antibodies to Malondialdehyde Conjugated With Albumin Are Negatively Associated With Cardiovascular Disease Among 60-Year-Olds.

BACKGROUND: Malondialdehyde (MDA) is generated during lipid peroxidation as in oxidized low-density lipoprotein, but antibodies against oxidized low-density lipoprotein show variable results in clinical studies. We therefore studied the risk of cardiovascular disease (CVD) associated with IgM antibodies against MDA conjugated with human albumin (anti-MDA). METHODS AND RESULTS: In a 5- to 7-year follow-up of 60-year-old men and women from Stockholm County previously screened for cardiovascular risk factors (2039 men, 2193 women), 209 incident CVD cases (defined as new events of coronary heart disease, fatal and nonfatal myocardial infarction, ischemic stroke, and hospitalization for angina pectoris) and 620 age- and sex-matched controls were tested for IgM anti-MDA by ELISA. Antibody peptide/protein characterization was done using a proteomics de novo sequencing approach. After adjustment for smoking, body-mass index, type 2 diabetes mellitus, hyperlipidemia, and hypertension, an increased CVD risk was observed in the low IgM anti-MDA percentiles (below 10th and 25th) (odds ratio and 95% CI: 2.0; 1.19-3.36 and 1.67; 1.16-2.41, respectively). Anti-MDA above the 66th percentile was associated with a decreased CVD risk (odds ratio 0.68; CI: 0.48-0.98). After stratification by sex, associations were only present among men. IgM anti-MDA levels were lower among cases (median [interquartile range]: 141.0 [112.7-164.3] versus 147.4 [123.5-169.6]; P=0.0177), even more so among men (130.6 [107.7-155.3] versus 143.0 [120.1-165.2]; P=0.001). The IgM anti-MDA variable region profiles are distinctly different and also more homologous in their content (correlates strongly with fewer peptides) than control antibodies (not binding MDA). CONCLUSIONS: IgM anti-MDA is a protection marker for CVD. This finding could have diagnostic and therapeutic implications.

Aberrant Niche Signaling in the Etiopathogenesis of Ulcerative Colitis.

BACKGROUND: Primary colonic epithelial defects leading to inflammatory responses are considered central to the development of ulcerative colitis (UC). However, a systematic analysis of various colonic subcompartments in the pathogenesis of UC before inflammation remains elusive. Here, we explored changes in colonic subcompartments and their associated niche signals in patient mucosal biopsies and in an animal model of colitis. METHODS: Analysis of mucosal biopsies obtained from uninvolved and involved regions of patients with UC and Crohn's disease was performed and compared with normal subjects. Temporal analysis of colonic subcompartments was performed in mice administered with 5% dextran sodium sulphate. Phenotypic enumeration of the crypt subcompartment was complemented with flow cytometric analysis. Members of Notch and Wnt signaling pathways were analyzed by molecular, biochemical, and colocalization studies. RESULTS: Phenotypic enumeration of colonocytes' subcompartments from patients revealed significant alterations of the lower crypt, enriched in stem cell and progenitors, independent of inflammation. These changes, unique to UC, were confirmed by immunohistochemistry and molecular analysis. In parallel, a defect in proliferation and Muc2 synthesis was observed. Animal data before inflammation recapitulated human studies. Mechanistic studies revealed that changes in signaling through Wnt primarily affected colonic stem cells, whereas Notch affected progenitor function. CONCLUSIONS: Our results thus provide new insights into the development of inflammation and relapse in UC and suggest that the stem cell niche in the colon may influence pathogenesis of the disease.

Oxidized but not native cardiolipin has pro-inflammatory effects, which are inhibited by Annexin A5.

OBJECTIVE: Cardiolipin (CL) is a phospholipid with an unusual dimeric structure containing four double-bonds and is easily oxidized. CL is present in mitochondria. Here we explored potential pro-inflammatory properties implicated in cardiovascular disease (CVD): activation of endothelial cells, 5-lipoxygenase (5-LOX) and leukotriene B4 (LTB4), by oxidized CL (oxCL) and inhibitory effects of Annexin A5, an antithrombotic and antiinflammatory plasma protein. METHODS: In monocytes/macrophages and neutrophils, calcium mobilization was monitored spectrophotometrically with Fura-2 and synthesis of LTB4 was analyzed by EIA. Expression of adhesion molecules on endothelial cells was studied by FACScan. Binding of Annexin A5 were analyzed by ELISA. The mRNA expression of 5-LOX and cyclooxygenase-2 was assessed by Real-Time PCR. RESULTS: We demonstrate that oxCL but not its non-oxidized counterpart CL induces biosynthesis of LTB4 and increases intracellular concentrations of calcium in monocytes/macrophages and neutrophils. oxCL rather than CL selectively elevates gene expression of 5-LOX but not COX-2 in human macrophages. Furthermore, oxCL but not CL raises levels of adhesion molecules ICAM-1 and VCAM-1 in endothelial cells. Annexin A5 can bind oxCL to abolish all these oxCL-induced effects. CONCLUSIONS: oxCL may promote inflammation and related diseases especially in conditions involving unresolved apoptosis and necrosis, such as atherosclerosis, where free oxCL is likely to be released from liberated mitochondria. Increased intracellular calcium could activate 5-LOX to produce Leukotriene B4 (LTB4). Annexin A5 inhibits the pro-inflammatory effects of oxCL and its potential therapeutic use when oxCL is implicated in inflammation could be of interest.