Metalloproteinases shed TREM-1 ectodomain from lipopolysaccharide-stimulated human monocytes

Triggering receptors expressed on myeloid cell (TREM) proteins are a family of cell surface receptors that participate in diverse cellular processes such as inflammation, coagulation, and bone homeostasis. TREM-1, in particular, is expressed on neutrophils and monocytes and is a potent amplifier of inflammatory responses. LPS and other microbial products induce up-regulation of cell surface-localized TREM-1 and the release of its soluble form, sTREM-1. Two hypotheses have been advanced to explain the origin of sTREM-1: alternative splicing of TREM-1 mRNA and proteolytic cleavage(s) of mature, membrane-anchored TREM-1. In this report, we present conclusive evidence in favor of the proteolytic mechanism of sTREM-1 generation. No alternative splicing forms of TREM-1 were detected in monocytes/macrophages. Besides, metalloproteinase inhibitors increased the stability of TREM-1 at the cell surface while significantly reducing sTREM-1 release in cultures of LPS-challenged human monocytes and neutrophils. We conclude that metalloproteinases are responsible for shedding of the TREM-1 ectodomain through proteolytic cleavage of its long juxtamembrane linker.     

Differential pattern of cell-surface and soluble TREM-1 between sepsis and SIRS

ObjectiveTriggering receptor expressed on myeloid cells-1 (TREM-1) was reported to play a key roll in amplification of production of inflammatory cytokines. TREM-1 is suggested to be a specific biomarker for sepsis for this reason, but the clinical significance of TREM-1 has not been elucidated. We investigated TREM-1 expression on the cell-surface, and plasma levels of soluble TREM-1 (sTREM-1) in patients with non-infectious systemic inflammatory response syndrome (SIRS) and sepsis admitted to the ICU.MethodsThirty-five patients with SIRS and 21 patients with sepsis admitted to ICU were subjected to the study. TREM-1 expressions on the surfaces of monocytes and neutrophils were measured by flow cytometry. Plasma sTREM-1 level and serum interleukin (IL)-6 level were measured.ResultsSeptic patients had decreased TREM-1 expression, clearly on neutrophils or to a lesser extent on monocyte compared to SIRS patients on ICU admission (neutrophils p < 0.001, monocyte p < 0.05). TREM-1 expression on neutrophils had a significant inverse correlation with serum IL-6 level (r = ?0.64, p < 0.0001). Plasma sTREM-1 level in septic patients was significantly higher than that in SIRS patients (p < 0.05). Plasma sTREM-1 level positively correlated with severity score and non-survivors had increased plasma sTREM-1 level compared to survivors in all SIRS/sepsis patients (p < 0.05).ConclusionsPatients with sepsis had increased soluble TREM-1 and decreased TREM-1 expression on neutrophil compared to SIRS patients. sTREM-1 may be useful to evaluate disease severity and outcome of patients with SIRS or sepsis.     

Cultured epithelial cells from patients with cystic fibrosis have an increased expression of the 14 kDa Ca2(+)-binding protein CFA

The Cystic Fibrosis antigen (CFA) is a 14 kDa. Ca2(+)-binding protein known to be expressed in cells of myeloid origin during normal cell differentiation. CFA serum levels are elevated in Cystic Fibrosis (CF) patients and heterozygotes. We examined the expression of CFA in different cultured epithelial cells from controls and patients with CF. The steady state level of CFA was in general higher in epithelial cells from CF patients compared to control cells and was found to increase during cell aging. The latter difference could be attributed to an increased rate of CFA synthesis rather than to an impairment of CFA degradation or secretion, as shown by pulse chase experiments.     

Lipopolysaccharide-induced up-regulation of triggering receptor expressed on myeloid cells-1 expression on macrophages is regulated by endogenous prostaglandin E2

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a recently identified cell surface molecule that is expressed by neutrophils and monocytes. TREM-1 expression is modulated by various ligands for TLRs in vitro and in vivo. However, the influence of PGE(2), a potential mediator of inflammation, on TREM-1 expression has not been elucidated. In this study, we examined the effects of PGE(2) on LPS-induced TREM-1 expression by resident murine peritoneal macrophages (RPM) and human PBMC. PGE(2) significantly induced murine TREM-1 (mTREM-1) expression by RPM. Up-regulation of TREM-1 expression was specific to PGE(2) among arachidonic acid metabolites, while ligands for chemoattractant receptor-homologous molecule expressed on Th2 cells and the thomboxane-like prostanoid receptor failed to induce mTREM-1 expression. PGE(2) also increased expression of the soluble form of TREM-1 by PBMC. LPS-induced TREM-1 expression was regulated by endogenous PGE(2) especially in late phase (>2 h after stimulation), because cyclooxygenase-1 and -2 inhibitors abolished this effect at that points. A synthetic EP4 agonist and 8-Br-cAMP also enhanced mTREM-1 expression by RPM. Furthermore, protein kinase A, PI3K, and p38 MAPK inhibitors prevented PGE(2)-induced mTREM-1 expression by RPM. Activation of TREM-1 expressed on PGE(2)-pretreated PBMC by an agonistic TREM-1 mAb significantly enhanced the production of IL-8 and TNF-alpha. These findings indicate that LPS-induced TREM-1 expression on macrophages is mediated, at least partly, by endogenous PGE(2) followed by EP4 and cAMP, protein kinase A, p38 MAPK, and PI3K-mediated signaling. Regulation of TREM-1 and the soluble form of TREM-1 expression by PGE(2) may modulate the inflammatory response to microbial pathogens.     

cAMP dependent chloride conductance is not different in cystic fibrosis fibroblasts

A chloride conductive pathway has been demonstrated in human skin fibroblasts and a defective cAMP dependent activation of this conductance in Cystic Fibrosis (CF) fibroblasts has been also reported. Chloride transport by the same reported method was studied in normal and CF skin fibroblasts. The stimulation of this pathway was not obtained consistently by the addition of dibutyryl cAMP. The addition of prostaglandin E1 (PGE1) increased the intracellular [cAMP] but did not increase the conductivity of the pathway consistently. Neither the basal nor the dibutyryl cAMP or the PGE1 stimulated chloride conductance differed significantly in CF fibroblasts.     

Proteomics investigation of human platelets in healthy donors and cystic fibrosis patients by shotgun nUPLC-MSE and 2DE: a comparative study

Platelets are of pathophysiological relevance in haemostasis, wound repair, inflammation and cardiovascular disease. We have shown that human platelets express a biologically active Cystic Fibrosis Transmembrane Conductance Regulator, which is dysfunctional in Cystic Fibrosis (CF) patients, and regulate platelet responses related to inflammation and its resolution. In order to further elucidate platelet involvement in CF inflammation, we pursued a comparative proteomic analysis of cells from healthy donors and CF patients, in association with a non-supervised comparative analysis of the Gene Ontology. Our results, showing changes in the integrin signalling in CF, support a pro-inflammatory profile of CF platelets.     

Functional genomics of silencing TREM-1 on TLR4 signaling in macrophages

Triggering receptor expressed on myeloid cells 1 (TREM-1) is a recently discovered molecule that is expressed on the cell surface of monocytes and neutrophils. Engagement of TREM-1 triggers synthesis of proinflammatory cytokines in response to microbes, but the extent and mechanism by which TREM-1 modulates the inflammatory response is poorly defined. In the present study, we investigated the functional effects of blocking TREM-1 on the Toll-like receptor (TLR)4-mediated signaling pathway in macrophages. By transfecting cells with small hairpin interfering RNA molecules to TREM-1 (shRNA), we confirmed that TREM-1 mRNA and protein expression was greatly attenuated in RAW cells in response to treatment with LPS. PCR array for genes related to or activated by the TLR pathway revealed that although the expression of TLR4 itself was not significantly altered by silencing of TREM-1, expression of several genes, including MyD88, CD14, IkappaBalpha, IL-1beta, MCP-1, and IL-10 was significantly attenuated in the TREM-1 knockdown cells in response to treatment with LPS. These data indicate that expression of TREM-1 modulates the TLR signaling in macrophages by altering the expression of both adaptor and effector proteins that are critical to the endotoxin response.     

Mechanisms of insulin resistance in cystic fibrosis

Cystic fibrosis (CF) is associated with a high incidence of diabetes. Studies evaluating causes of CF-related diabetes (CFRD) have consistently documented decreased insulin secretion. In patients with CFRD, insulin sensitivity has been documented to be decreased, but controversy exists in patients with normal or impaired glucose tolerance (IGT). We undertook this study 1) to reexplore insulin sensitivity in patients with IGT and 2) to evaluate potential mechanisms of insulin resistance in CF, including GLUT-4 translocation, elevation of serum cytokines, and free fatty acid (FFA) levels. We recruited nine CF subjects with impaired glucose tolerance (IGTCF) and nine age-, gender-, and body mass index-matched control volunteers. Each underwent a hyperinsulinemic euglycemic clamp (200 mU. m(-2). min(-1)) to measure insulin sensitivity. A muscle biopsy was obtained at maximal insulin stimulation for measure of GLUT-4 translocation with sucrose gradients. An oral glucose tolerance test and National Institutes of Health (NIH) clinical status scores were measured in all volunteers. We also measured tumor necrosis factor (TNF)-alpha levels and FFA in all subjects. Additionally, we report the results of TNF-alpha and FFA in 32 CF patients previously studied by our group. Results were that glucose disposal rate (GDR) was significantly lower in the CFIGT subjects than in controls, indicative of impaired insulin action. GLUT-4 translocation was impaired in CF and correlated with GDR. TNF-alpha levels were higher in all CF subjects than in controls and correlated with GDR. There was no difference in FFA between CF and control subjects. Modified NIH clinical status scores were inversely correlated with GDR and TNF-alpha levels. We conclude that IGTCF patients have decreased peripheral insulin sensitivity. Mechanisms include elevation of TNF-alpha and impaired translocation of GLUT-4.     

Monocytes from Depressed Patients Display an Altered Pattern of Response to Endotoxin Challenge

It is now well established that major depression is accompanied and characterized by altered responses of the immune-inflammatory system. In this study we investigated the pro-inflammatory activation of monocytes isolated from depressed patients as a parameter not influenced by such confounds as the time of day, the nutritional and exercise status or the age and gender of patients. Monocytes from depressed patients and from healthy controls were isolated in vitro; after 24-h incubation under basal conditions, cells were exposed for 24-h to 100 ng/ml of endotoxin (bacterial lipopolysaccharide, LPS). We found that monocytes from drug-free depressed patients and controls release the same amounts of prostaglandin E2 (PGE2) under basal conditions, whereas monocytes from patients are dramatically less reactive to LPS (8.62-fold increase vs previous 24 hrs) compared to healthy controls (123.3-fold increase vs previous 24 hrs). Such blunted prostanoid production was paralleled by a reduction in COX-2 gene expression, whereas other pro-inflammatory mediators, namely interleukin-1β (IL-1 β) and -6 (IL-6) showed a trend to increased gene expression. The above changes were not associated to increased levels of circulating glucocorticoids. After 8 months of antidepressive drug treatment, the increase in PGE2 production after the endotoxin challenge was partially restored, whereas the increase in IL-1 β and -6 levels observed at baseline was completely abolished. In conclusion, our findings show that the reactivity of monocytes from depressed patients might be considered as a marker of the immune-inflammatory disorders associated to depression, although the lack of paired healthy controls at follow-up does not allow to conclude that monocyte reactivity to endotoxin is also a marker of treatment outcome.     

A Biomarker Panel (Bioscore) Incorporating Monocytic Surface and Soluble TREM-1 Has High Discriminative Value for Ventilator-Associated Pneumonia: A Prospective Observational Study

Introduction

Ventilator-associated pneumonia (VAP) increases mortality in critical illness. However, clinical diagnostic uncertainty persists. We hypothesised that measuring cell-surface and soluble inflammatory markers, incorporating Triggering Receptor Expressed by Myeloid cells (TREM)-1, would improve diagnostic accuracy.

Methods

A single centre prospective observational study, set in a University Hospital medical-surgical intensive Care unit, recruited 91 patients into 3 groups: 27 patients with VAP, 33 ventilated controls without evidence of pulmonary sepsis (non-VAP), and 31 non-ventilated controls (NVC), without clinical infection, attending for bronchoscopy. Paired samples of Bronchiolo-alveolar lavage fluid (BALF) and blood from each subject were analysed for putative biomarkers of infection: Cellular (TREM-1, CD11b and CD62L) and soluble (IL-1β, IL-6, IL-8, sTREM-1, Procalcitonin). Expression of cellular markers on monocytes and neutrophils were measured by flow cytometry. Soluble inflammatory markers were determined by ELISA. A biomarker panel (‘Bioscore’), was constructed, tested and validated, using Fisher’s discriminant function analysis, to assess its value in distinguishing VAP from non VAP.

Results

The expression of TREM-1 on monocytes (mTREM-1) and neutrophils (nTREM-1) and concentrations of IL-1β, IL-8, and sTREM-1 in BALF were significantly higher in VAP compared with non-VAP and NVC (p<0.001). The BALF/blood mTREM-1 was significantly higher in VAP patients compared to non-VAP and NVC (0.8 v 0.4 v 0.3 p<0.001). A seven marker Bioscore (BALF/blood ratio mTREM-1 and mCD11b, BALF sTREM-1, IL-8 and IL-1β, and serum CRP and IL-6) correctly identified 88.9% of VAP cases and 100% of non-VAP cases.

Conclusion

A 7-marker bioscore, incorporating cellular and soluble TREM-1, accurately discriminates VAP from non-pulmonary infection.     

Prognosis of community acquired pneumonia (CAP): value of triggering receptor expressed on myeloid cells-1 (TREM-1) and other mediators of the inflammatory response

TREM-1 is an activating receptor expressed on the surface of neutrophils and mature monocytes when stimulated by bacteria or fungi, leading to amplification of the inflammatory response. Our objective is to analyze the prognostic value of serum sTREM-1 levels and other mediators of the inflammatory response, in patients hospitalized for CAP, and to compare its prognostic value with those of advanced age, pneumonia severity scores, Charlson index, nutritional status and severity of sepsis. METHODS: We included 226 patients with CAP, 145 males and 81 females, median age of 74 years. The following tests were performed: arterial blood gases and chest radiography, nutritional assessment, assessment of the severity of the sepsis, Pneumonia Severity Index (PSI) and CURB-65, and mediators of inflammation: TNF alfa, IL-6, IL-10, IL-1ra, LBP, sCD14, CRP, and sTREM-1. Mortality during admittance was defined as the sole end point. RESULTS: Twenty-eight of the two-hundred and twenty-six patients died (12.4%). On univariate analysis advanced age, dehydration, increased Na, low BMI, handgrip strength, serum albumin, prealbumin, IGF-1, lymphocyte count, conscious drowsiness, tachypnea, decreased PaO2, hypotension, creatinine, ASAT, LDH, severity of sepsis, a high PSI or CURB65, TNFalpha, IL-6, IL-10, IL-1ra, and sTREM-1 were related to mortality. Variables with an independent value were IGF-1, CURB-65, TREM-1, advanced age and IL-6. CONCLUSIONS: This study confirms the usefulness of TREM-1 in the diagnosis and prognosis of patients with CAP, which is independent of advanced age, other inflammation markers such as IL-6, severity index for CAP such as CURB-65 or PSI, severity of sepsis and nutritional status including IGF-1.     

Identification of Predictive Early Biomarkers for Sterile-SIRS after Cardiovascular Surgery

Systemic inflammatory response syndrome (SIRS) is a common complication after cardiovascular surgery that in severe cases can lead to multiple organ dysfunction syndrome and even death. We therefore set out to identify reliable early biomarkers for SIRS in a prospective small patient study for timely intervention. 21 Patients scheduled for planned cardiovascular surgery were recruited in the study, monitored for signs of SIRS and blood samples were taken to investigate biomarkers at pre-assigned time points: day of admission, start of surgery, end of surgery, days 1, 2, 3, 5 and 8 post surgery. Stored plasma and cryopreserved blood samples were analyzed for cytokine expression (IL1β, IL2, IL6, IL8, IL10, TNFα, IFNγ), other pro-inflammatory markers (sCD163, sTREM-1, ESM-1) and response to endotoxin. Acute phase proteins CRP, PCT and pro-inflammatory cytokines IL6 and IL8 were significantly increased (p<0.001) at the end of surgery in all patients but could not distinguish between groups. Normalization of samples revealed significant increases in IL1β changes (p<0.05) and decreased responses to endotoxin (p<0.01) in the SIRS group at the end of surgery. Soluble TREM-1 plasma concentrations were significantly increased in patients with SIRS (p<0.01). This small scale patient study could show that common sepsis markers PCT, CRP, IL6 and TNFα had low predictive value for early diagnosis of SIRS after cardiovascular surgery. A combination of normalized IL1β plasma levels, responses to endotoxin and soluble TREM-1 plasma concentrations at the end of surgery are predictive markers of SIRS development in this small scale study and could act as an indicator for starting early therapeutic interventions.     

Impact of CFTR DeltaF508 mutation on prostaglandin E2 production and type IIA phospholipase A2 expression by pulmonary epithelial cells

Cystic fibrosis (CF) is characterized by an exacerbated inflammatory pulmonary response with excessive production of inflammatory mediators. We investigated here the impact of cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction on prostaglandin E2 (PGE2) production and type IIA secreted phospholipase A2 (sPLA2-IIA) expression. We show that both resting and LPS-stimulated human respiratory epithelial cell line bearing DeltaF508 mutation on CFTR (CF cells) released more PGE2 than control cell line. This was accompanied by enhanced expression and activity of cyclooxygenase-2 in CF cells. PGE2 release was attenuated after experimentally induced retrafficking of the DeltaF508-CFTR at the plasma membrane. sPLA2-IIA expression occurred at higher levels in CF cells than in control cells and was enhanced by LPS and PGE2. Suppression of PGE2 synthesis by aspirin led to an inhibition of LPS-induced sPLA2-IIA expression. Higher activation of NF-kappaB was observed in CF cells compared with control cells and was enhanced by LPS. However, addition of PGE2 or aspirin had no effect on NF-kappaB activation. LPS-induced sPLA2-IIA expression was reduced by an NF-kappaB inhibitor. We suggest that the lack of the CFTR in the plasma membrane results in a PGE2 overproduction and an enhanced sPLA2-IIA expression. This expression is upregulated by NF-kappaB and amplified by PGE2 via a unidentified signaling pathway.     

Soluble TREM-1, as a new ligand for the membrane receptor Robo2, promotes hepatic stellate cells activation and liver fibrosis

Triggering receptor expressed on myeloid cells-1 (TREM-1) exists in two forms: a transmembrane form and a soluble form (sTREM-1). The levels of sTREM-1 are elevated in supernatants of activated HSCs. However, the role of sTREM-1 in HSC activation and liver fibrosis remains undefined. Previous studies have primarily focused on the transmembrane form of TREM-1; we innovatively observed the function of sTREM-1 as a ligand in liver fibrosis and screened its receptor. Here, recombinant sTREM-1 was used as a stimulator which induced HSC activation and further aggravated liver fibrosis. Then, screening for sTREM-1 interacting membrane receptors was performed using pull-down assay followed by mass spectrometry, and the membrane receptor roundabout guidance receptor 2 (Robo2) was identified as a candidate receptor for sTREM-1. The interaction between sTREM-1 and Robo2 was verified by pull-down and immunofluorescence. The role of Robo2 on sTREM-1-induced HSC activation and its downstream signal pathways was assessed by knockdown of Robo2 in LX-2 cells. Furthermore, HSC-specific knockdown of Robo2 was achieved in a mouse model of liver fibrosis by using a recombinant adeno-associated virus (AAV) vector to confirm the role of the receptor, and we proved that Robo2 knockdown inhibited the activation of HSC and liver fibrosis, which also led to the inactivation of Smad2/3 and PI3K/Akt pathways in sTREM-1-induced HSC activation and liver fibrosis. In conclusion, sTREM-1 acts as a new ligand of Robo2; the binding of sTREM-1 to Robo2 initiates the activation of the downstream Smad2/3 and PI3K/Akt signalling pathways, thereby promoting HSC activation and liver fibrosis.     

Early bronchial inflammation in cystic fibrosis

Cystic fibrosis (CF) is the most common genetic autosomal recessive disease in caucasian north-american and european populations. The CF gene codes for a transmembrane glycoprotein called CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), a chloride channel which regulates the luminal secretion of chloride and the active ion and water transport in the airway epithelial cells. Mutations of the CF gene lead to a dysregulation of chloride and sodium channel associated to airway mucus dehydration, neutrophil-dominated airway inflammation and chronic infection responsible for the morbidity and mortality of CF patients. Although a high number of studies has been devoted to the CFTR pleiotropic functions, the chronology of the physiopathological events leading to the airway inflammation linked to mutations of the CF gene is still an open question. The issue of whether airway inflammation takes place before infection or is a consequence of infection during CF pathogenesis is still controversial. It has been recently reported that in broncho-alveolar lavages collected in CF infants, there is an increased level of interleukin IL-8 and abnormal low level of IL-10. The decreased IL-10 production has been confirmed in peripheral blood monocytes as well as in airway cell lines. Under basal conditions, the increased expression of the pro-inflammatory IL-8 cytokine has also been recently observed in the airway liquid secreted by CF na?ve humanized airway xenografts and in the supernatant culture of CF human airway epithelial cells. These results suggest that CFTR dysfunction may result in a constitutive pro-inflammatory vs anti-inflammatory imbalance in CF disease. Recent data from the literature suggest that the failure of chloride transport, the maturation defect and mistraffricking of mutated CFTR, lead to its accumulation in the endoplasmic reticulum and activation of NF-kappa B, responsible for the imbalance in the CF airway cell cytokine production.     

Elevated miR-155 promotes inflammation in cystic fibrosis by driving hyperexpression of interleukin-8

Cystic Fibrosis (CF) is characterized by a massive proinflammatory phenotype in the lung arising from profound expression of inflammatory genes, including interleukin-8 (IL-8). We have previously reported that IL-8 mRNA is stabilized in CF lung epithelial cells, resulting in concomitant hyperexpression of IL-8 protein. However, the mechanistic link between mutations in CFTR and acquisition of the proinflammatory phenotype in the CF airway has remained elusive. We hypothesized that specific microRNAs (miRNAs) might mediate this linkage. To identify the potential link, we screened an miRNA library for differential expression in ΔF508-CFTR and wild type CFTR lung epithelial cell lines. Of 22 differentially and significantly expressed miRNAs, we found that expression of miR-155 was more than 5-fold elevated in CF IB3-1 lung epithelial cells in culture, compared with control IB3-1/S9 cells. Clinically, miR-155 was also highly expressed in CF lung epithelial cells and circulating CF neutrophils biopsied from CF patients. We report here that high levels of miR-155 specifically reduced levels of SHIP1, thereby promoting PI3K/Akt activation. However, overexpressing SHIP1 or inhibition of PI3K in CF cells suppressed IL-8 expression. Finally, we found that phospho-Akt levels were elevated in CF lung epithelial cells and were specifically lowered by either antagomir-155 or elevated expression of SHIP1. We therefore suggest that elevated miR-155 contributes to the proinflammatory expression of IL-8 in CF lung epithelial cells by lowering SHIP1 expression and thereby activating the PI3K/Akt signaling pathway. These data suggest that miR-155 may play an important role in the activation of IL-8-dependent inflammation in CF.     

CFTR expression analysis in human nasal epithelial cells by flow cytometry

Rationale

Unbiased approaches that study aberrant protein expression in primary airway epithelial cells at single cell level may profoundly improve diagnosis and understanding of airway diseases. We here present a flow cytometric procedure to study CFTR expression in human primary nasal epithelial cells from patients with Cystic Fibrosis (CF). Our novel approach may be important in monitoring of therapeutic responses, and better understanding of CF disease at the molecular level.

Objectives

Validation of a panel of CFTR-directed monoclonal antibodies for flow cytometry and CFTR expression analysis in nasal epithelial cells from healthy controls and CF patients.

Methods

We analyzed CFTR expression in primary nasal epithelial cells at single cell level using flow cytometry. Nasal cells were stained for pan-Cytokeratin, E cadherin, and CD45 (to discriminate epithelial cells and leukocytes) in combination with intracellular staining of CFTR. Healthy individuals and CF patients were compared.

Measurements and Main Results

We observed various cellular populations present in nasal brushings that expressed CFTR protein at different levels. Our data indicated that CF patients homozygous for F508del express varying levels of CFTR protein in nasal epithelial cells, although at a lower level than healthy controls.

Conclusion

CFTR protein is expressed in CF patients harboring F508del mutations but at lower levels than in healthy controls. Multicolor flow cytometry of nasal cells is a relatively simple procedure to analyze the composition of cellular subpopulations and protein expression at single cell level.