Autocrine vitamin D signaling switches off pro-inflammatory programs of TH1 cells.

The molecular mechanisms governing orderly shutdown and retraction of CD4+ type 1 helper T (TH1) cell responses remain poorly understood. Here we show that complement triggers contraction of TH1 responses by inducing intrinsic expression of the vitamin D (VitD) receptor and the VitD-activating enzyme CYP27B1, permitting T cells to both activate and respond to VitD. VitD then initiated the transition from pro-inflammatory interferon-γ+ TH1 cells to suppressive interleukin-10+ cells. This process was primed by dynamic changes in the epigenetic landscape of CD4+ T cells, generating super-enhancers and recruiting several transcription factors, notably c-JUN, STAT3 and BACH2, which together with VitD receptor shaped the transcriptional response to VitD. Accordingly, VitD did not induce interleukin-10 expression in cells with dysfunctional BACH2 or STAT3. Bronchoalveolar lavage fluid CD4+ T cells of patients with COVID-19 were TH1-skewed and showed de-repression of genes downregulated by VitD, from either lack of substrate (VitD deficiency) and/or abnormal regulation of this system.

Diapedesis-Induced Integrin Signaling via LFA-1 Facilitates Tissue Immunity by Inducing Intrinsic Complement C3 Expression in Immune Cells.

Intrinsic complement C3 activity is integral to human T helper type 1 (Th1) and cytotoxic T cell responses. Increased or decreased intracellular C3 results in autoimmunity and infections, respectively. The mechanisms regulating intracellular C3 expression remain undefined. We identified complement, including C3, as among the most significantly enriched biological pathway in tissue-occupying cells. We generated C3-reporter mice and confirmed that C3 expression was a defining feature of tissue-immune cells, including T cells and monocytes, occurred during transendothelial diapedesis, and depended on integrin lymphocyte-function-associated antigen 1 (LFA-1) signals. Immune cells from patients with leukocyte adhesion deficiency type 1 (LAD-1) had reduced C3 transcripts and diminished effector activities, which could be rescued proportionally by intracellular C3 provision. Conversely, increased C3 expression by T cells from arthritis patients correlated with disease severity. Our study defines integrins as key controllers of intracellular complement, demonstrates that perturbations in the LFA-1-C3-axis contribute to primary immunodeficiency, and identifies intracellular C3 as biomarker of severity in autoimmunity.

NOS2 and S-nitrosothiol signaling induces DNA hypomethylation and LINE-1 retrotransposon expression.

Inducible nitric oxide synthase (NOS2) produces high local concentrations of nitric oxide (NO), and its expression is associated with inflammation, cellular stress signals, and cellular transformation. Additionally, NOS2 expression results in aggressive cancer cell phenotypes and is correlated with poor outcomes in patients with breast cancer. DNA hypomethylation, especially of noncoding repeat elements, is an early event in carcinogenesis and is a common feature of cancer cells. In addition to altered gene expression, DNA hypomethylation results in genomic instability via retrotransposon activation. Here, we show that NOS2 expression and associated NO signaling results in substantial DNA hypomethylation in human cell lines by inducing the degradation of DNA (cytosine-5)­methyltransferase 1 (DNMT1) protein. Similarly, NOS2 expression levels were correlated with decreased DNA methylation in human breast tumors. NOS2 expression and NO signaling also resulted in long interspersed noncoding element 1 (LINE-1) retrotransposon hypomethylation, expression, and DNA damage. DNMT1 degradation was mediated by an NO/p38-MAPK/lysine acetyltransferase 5­dependent mechanism. Furthermore, we show that this mechanism is required for NO-mediated epithelial transformation. Therefore, we conclude that NOS2 and NO signaling results in DNA damage and malignant cellular transformation via an epigenetic mechanism.

Complement Regulates Nutrient Influx and Metabolic Reprogramming during Th1 Cell Responses.

Expansion and acquisition of Th1 cell effector function requires metabolic reprogramming; however, the signals instructing these adaptations remain poorly defined. Here we found that in activated human T cells, autocrine stimulation of the complement receptor CD46, and specifically its intracellular domain CYT-1, was required for induction of the amino acid (AA) transporter LAT1 and enhanced expression of the glucose transporter GLUT1. Furthermore, CD46 activation simultaneously drove expression of LAMTOR5, which mediated assembly of the AA-sensing Ragulator-Rag-mTORC1 complex and increased glycolysis and oxidative phosphorylation (OXPHOS), required for cytokine production. T cells from CD46-deficient patients, characterized by defective Th1 cell induction, failed to upregulate the molecular components of this metabolic program as well as glycolysis and OXPHOS, but IFN-γ production could be reinstated by retrovirus-mediated CD46-CYT-1 expression. These data establish a critical link between the complement system and immunometabolic adaptations driving human CD4(+) T cell effector function.

Defective STAT5 Activation and Aberrant Expression of BCL6 in Naive CD4 T Cells Enhances Follicular Th Cell-like Differentiation in Patients with Granulomatosis with Polyangiitis.

Granulomatosis with polyangiitis (GPA) is a potentially fatal small vessel vasculitis of unknown etiology, characterized by anti-neutrophil cytoplasmic autoantibodies, chronic inflammation, and granulomatous tissue damage. T cell dysregulation, comprising decreased regulatory T cell function and increased circulating effector memory follicular Th cells (TFH), is strongly associated with disease pathogenesis, but the mechanisms driving these observations are unknown. We undertook transcriptomic and functional analysis of naive CD4 T cells from patients with GPA to identify underlying functional defects that could manifest in the pathogenic profiles observed in GPA. Gene expression studies revealed a dysregulation of the IL-2 receptor ß/JAK-STAT signaling pathway and higher expression of BCL6 and BCL6-regulated genes in GPA naive CD4 T cells. IL-2-induced STAT5 activation in GPA naive CD4 T cells was decreased, whereas STAT3 activation by IL-6 and IL-2 was unperturbed. Consistently, BCL6 expression was sustained following T cell activation of GPA naive CD4 T cells and in vitro TFH differentiation of these cells resulted in significant increases in the production TFH-related cytokines IL-21 and IL-6. Thus, naive CD4 T cells are dysregulated in patients with GPA, resulting from an imbalance in signaling equilibrium and transcriptional changes that drives the skewed pathogenic CD4 effector immune response in GPA.

IKZF3/Aiolos Is Associated with but Not Sufficient for the Expression of IL-10 by CD4+ T Cells.

The expression of anti-inflammatory IL-10 by CD4+ T cells is indispensable for immune homeostasis, as it allows T cells to moderate their effector function. We previously showed that TNF-α blockade during T cell stimulation in CD4+ T cell/monocyte cocultures resulted in maintenance of IL-10-producing T cells and identified IKZF3 as a putative regulator of IL-10. In this study, we tested the hypothesis that IKZF3 is a transcriptional regulator of IL-10 using a human CD4+ T cell-only culture system. IL-10+ CD4+ T cells expressed the highest levels of IKZF3 both ex vivo and after activation compared with IL-10-CD4+ T cells. Pharmacological targeting of IKZF3 with the drug lenalidomide showed that IKZF3 is required for anti-CD3/CD28 mAb-mediated induction of IL-10 but is dispensable for ex vivo IL-10 expression. However, overexpression of IKZF3 was unable to upregulate IL-10 at the mRNA or protein level in CD4+ T cells and did not drive the transcription of the IL10 promoter or putative local enhancer constructs. Collectively, these data indicate that IKZF3 is associated with but not sufficient for IL-10 expression in CD4+ T cells.

Interleukin-22 orchestrates a pathological endoplasmic reticulum stress response transcriptional programme in colonic epithelial cells.

OBJECTIVE: The functional role of interleukin-22 (IL22) in chronic inflammation is controversial, and mechanistic insights into how it regulates target tissue are lacking. In this study, we evaluated the functional role of IL22 in chronic colitis and probed mechanisms of IL22-mediated regulation of colonic epithelial cells. DESIGN: To investigate the functional role of IL22 in chronic colitis and how it regulates colonic epithelial cells, we employed a three-dimentional mini-gut epithelial organoid system, in vivo disease models and transcriptomic datasets in human IBD. RESULTS: As well as inducing transcriptional modules implicated in antimicrobial responses, IL22 also coordinated an endoplasmic reticulum (ER) stress response transcriptional programme in colonic epithelial cells. In the colon of patients with active colonic Crohn's disease (CD), there was enrichment of IL22-responsive transcriptional modules and ER stress response modules. Strikingly, in an IL22-dependent model of chronic colitis, targeting IL22 alleviated colonic epithelial ER stress and attenuated colitis. Pharmacological modulation of the ER stress response similarly impacted the severity of colitis. In patients with colonic CD, antibody blockade of IL12p40, which simultaneously blocks IL12 and IL23, the key upstream regulator of IL22 production, alleviated the colonic epithelial ER stress response. CONCLUSIONS: Our data challenge perceptions of IL22 as a predominantly beneficial cytokine in IBD and provide novel insights into the molecular mechanisms of IL22-mediated pathogenicity in chronic colitis. Targeting IL22-regulated pathways and alleviating colonic epithelial ER stress may represent promising therapeutic strategies in patients with colitis. TRIAL REGISTRATION NUMBER: NCT02749630.

Correction of Defective T-Regulatory Cells From Patients With Crohn's Disease by Ex Vivo Ligation of Retinoic Acid Receptor-α.

BACKGROUND & AIMS: Crohn's disease (CD) is characterized by an imbalance of effector and regulatory T cells in the intestinal mucosa. The efficacy of anti-adhesion therapies led us to investigate whether impaired trafficking of T-regulatory (Treg) cells contributes to the pathogenesis of CD. We also investigated whether proper function could be restored to Treg cells by ex vivo expansion in the presence of factors that activate their regulatory activities. METHODS: We measured levels of the integrin α4ß7 on Treg cells isolated from peripheral blood or lamina propria of patients with CD and healthy individuals (controls). Treg cells were expanded ex vivo and incubated with rapamycin with or without agonists of the retinoic acid receptor-α (RARA), and their gene expression profiles were analyzed. We also studied the cells in cytokine challenge, suppression, and flow chamber assays and in SCID mice with human intestinal xenografts. RESULTS: We found that Treg cells from patients with CD express lower levels of the integrin α4ß7 than Treg cells from control patients. The pathway that regulates the expression of integrin subunit α is induced by retinoic acid (RA). Treg cells from patients with CD incubated with rapamycin and an agonist of RARA (RAR568) expressed high levels of integrin α4ß7, as well as CD62L and FOXP3, compared with cells incubated with rapamycin or rapamycin and all-trans retinoic acid. These Treg cells had increased suppressive activities in assays and migrated under conditions of shear flow; they did not produce inflammatory cytokines, and RAR568 had no effect on cell stability or lineage commitment. Fluorescently labeled Treg cells incubated with RAR568 were significantly more likely to traffic to intestinal xenografts than Treg cells expanded in control medium. CONCLUSIONS: Treg cells from patients with CD express lower levels of the integrin α4ß7 than Treg cells from control patients. Incubation of patients' ex vivo expanded Treg cells with rapamycin and an RARA agonist induced expression of α4ß7 and had suppressive and migratory activities in culture and in intestinal xenografts in mice. These cells might be developed for treatment of CD. ClinicalTrials.gov, Number: NCT03185000.

SOX2 Drives Bronchial Dysplasia in a Novel Organotypic Model of Early Human Squamous Lung Cancer.

RATIONALE: Improving the early detection and chemoprevention of lung cancer are key to improving outcomes. The pathobiology of early squamous lung cancer is poorly understood. We have shown that amplification of sex-determining region Y-box 2 (SOX2) is an early and consistent event in the pathogenesis of this disease, but its functional oncogenic potential remains uncertain. We tested the impact of deregulated SOX2 expression in a novel organotypic system that recreates the molecular and microenvironmental context in which squamous carcinogenesis occurs. OBJECTIVES: (1) To develop an in vitro model of bronchial dysplasia that recapitulates key molecular and phenotypic characteristics of the human disease; (2) to test the hypothesis that SOX2 deregulation is a key early event in the pathogenesis of bronchial dysplasia; and (3) to use the model for studies on pathogenesis and chemoprevention. METHODS: We engineered the inducible activation of oncogenes in immortalized bronchial epithelial cells. We used three-dimensional tissue culture to build an organotypic model of bronchial dysplasia. MEASUREMENTS AND MAIN RESULTS: We recapitulated human bronchial dysplasia in vitro. SOX2 deregulation drives dysplasia, and loss of tumor promoter 53 is a cooperating genetic event that potentiates the dysplastic phenotype. Deregulated SOX2 alters critical genes implicated in hallmarks of cancer progression. Targeted inhibition of AKT prevents the initiation of the dysplastic phenotype. CONCLUSIONS: In the appropriate genetic and microenvironmental context, acute deregulation of SOX2 drives bronchial dysplasia. This confirms its oncogenic potential in human cells and affords novel insights into the impact of SOX2 deregulation. This model can be used to test therapeutic agents aimed at chemoprevention.

Activation-Associated Accelerated Apoptosis of Memory B Cells in Critically Ill Patients With Sepsis.

OBJECTIVE: Sepsis is life-threatening organ dysfunction due to dysregulated host responses to infection. Current knowledge of human B-cell alterations in sepsis is sparse. We tested the hypothesis that B-cell loss in sepsis involves distinct subpopulations of B cells and investigated mechanisms of B-cell depletion. DESIGN: Prospective cohort study. SETTING: Critical care units. PATIENTS: Adult sepsis patients without any documented immune comorbidity. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: B-cell subsets were quantified by flow cytometry; annexin-V status identified apoptotic cells and phosphorylation of intracellular kinases identified activation status of B-cell subsets. B cell-specific survival ligand concentrations were measured. Gene expression in purified B cells was measured by microarray. Differences in messenger RNA abundance between sepsis and healthy controls were compared. Lymphopenia present in 74.2% of patients on admission day was associated with lower absolute B-cell counts (median [interquartile range], 0.133 [0.093-0.277] 10 cells/L) and selective depletion of memory B cells despite normal B cell survival ligand concentrations. Greater apoptotic depletion of class-switched and IgM memory cells was associated with phosphorylation of extracellular signal-regulated kinases, implying externally driven lymphocyte stress and activation-associated cell death. This inference is supported by gene expression profiles highlighting mitochondrial dysfunction and cell death pathways, with enriched intrinsic and extrinsic pathway apoptosis genes. CONCLUSIONS: Depletion of the memory B-cell compartment contributes to the immunosuppression induced by sepsis. Therapies targeted at reversing this immune memory depletion warrant further investigation.

Stress-activated Dendritic Cells (DC) Induce Dual Interleukin (IL)-15- and IL1ß-mediated Pathways, Which May Elicit CD4+ Memory T Cells and Interferon (IFN)-stimulated Genes.

The prevailing evidence suggests that immunological memory does not require antigenic re-stimulation but is maintained by low level tonic stimulation. We examined the hypothesis that stress agents contribute to tonic cellular activation and maintain immunological memory. Stimulation of monocyte-derived dendritic cells (DC) with stress agents elicits reactive oxygen species and HSP70. NFκB is activated, which up-regulates membrane-associated (ma) IL-15, caspase-1 and IL-1ß. Co-culture of stress-treated DC with mononuclear cells activates IL-15 and IL-1ß receptors on CD4(+) T cells, eliciting CD40L, proliferation, and up-regulation of CD45RO(+) memory T cells. The transcription factors Tbet(high) and RORγt are up-regulated, whereas FoxP3 is down-regulated, resulting in enhanced Th1 and Th17 expression and the corresponding cytokines. The interaction between maIL-15 expressed by DC and IL-15R on CD4(+) T cells results in one pathway and the corresponding cells expressing IL-1ß and IL1ßR as a second pathway. Importantly, inhibition studies with IL-15 antibodies and IL-1ßR inhibitor suggest that both pathways may be required for optimum CD4(+) CD45RO(+) memory T cell expression. Type 1 IFN expression in splenic CD11c DC of stress-treated mice demonstrated a significant increase of IFN-α in CD11c CD317(+) and CD8α(+) DC. Analysis of RNA in human CD4(+) memory T cells showed up-regulation of type 1 IFN-stimulated genes and inhibition with histone methyltransferase inhibitor. We suggest the paradigm that stress-induced tonic stimulation might be responsible for the robust persistence of the immune response in vaccination and that epigenetic changes are involved in maintaining CD4(+) T cell memory.

Human IL-31 is induced by IL-4 and promotes TH2-driven inflammation.

BACKGROUND: The pruritic cytokine IL-31 has been shown to be expressed by murine activated effector T Lymphocytes of a TH2 phenotype. Like IL-17 and IL-22, IL-31 is a tissue-signaling cytokine the receptor of which is mainly found on nonimmune cells. An overabundance of IL-31 has been shown in patients with atopic disorders, including dermatitis, as well as asthma, and therefore represents a promising drug target, although its regulation in the context of the human TH2 clusters is not yet known. OBJECTIVE: We sought to address the gene regulation of human IL-31 and to test whether IL-31 possesses a similar proallergic function as members of the human TH2 cytokine family, such as IL-4, IL-5, and IL-13. METHODS: Polyclonal and purified protein derivative of tuburculin-specific T-cell clones were generated. TH phenotype was determined, and IL-31 was measured by means of ELISA. Gene expression of primary bronchial epithelial cells treated with IL-31 was also measured. RESULTS: IL-31 was expressed by all of the TH2 clones and not by TH1, TH17, or TH22. This expression was dependent on autocrine IL-4 expression from these clones because it could be reduced if blocking antibodies to IL-4 were present. Interestingly, TH1 clones were able to express IL-31 if IL-4 was added to culture. This IL-31 expression was transient and did not affect the phenotype of the TH1 clones. IL-31 was able to induce proinflammatory genes, such as CCL2 and granulocyte colony-stimulating factor. CONCLUSION: IL-31 is not a TH2 cytokine in the classical sense but is likely to be expressed by a number of cells in an allergic situation in which IL-4 is present and possibly contribute to the allergic reaction.

Epigenetic Approaches to Identifying Asthma Endotypes.

The prevalence of asthma escalated rapidly in the late 20th century. In 2019, the World Health Organization estimated the global number of people affected by the condition to be approximately 260 million, causing 450,000 deaths during that year. While there have been advances in therapeutics with the emergence of biologics targeting T2-high asthma, there is still little clarity on the mechanisms underlying the origins of both the condition and all of its endotypes. Several biomarkers for particular asthma phenotypes have been documented. These are generally identified from transcriptomics and proteomics protocols and tend to be biased to T2-high phenotypes. In this review, we summarize some suggestions that analysis of epigenomes may provide alternative datasets that inform of broader asthma endotypes and might highlight pathways amenable for therapeutic intervention.

Identification of a transcription factor network regulating anti-TNF mediated IL10 expression in human CD4+ T cells.

CD4+ T cells are key players in immune-mediated inflammatory diseases (IMIDs) through the production of inflammatory mediators including tumour necrosis factor (TNF). Anti-TNF therapy has revolutionized the treatment of several IMIDs and we previously demonstrated that in vitro treatment of human CD4+ T cells with anti-TNF promotes anti-inflammatory IL-10 expression in multiple subpopulations of CD4+ T cells. Here we investigated the transcriptional mechanisms underlying the IL-10 induction by TNF-blockade in CD4+ T cells, isolated from PBMCs of healthy volunteers, stimulated in vitro for 3 days with anti-CD3/CD28 mAb in the absence or presence of anti-TNF. After culture, CD45RA+ cells were depleted before performing gene expression profiling and chromatin accessibility analysis. Gene expression analysis of CD45RA-CD4+ T cells showed a distinct anti-TNF specific gene signature of 183 genes (q-value < 0.05). Pathway enrichment analysis of differentially expressed genes revealed multiple pathways related to cytokine signalling and regulation of cytokine production; in particular, IL10 was the most upregulated gene by anti-TNF, while the proinflammatory cytokines and chemokines IFNG, IL9, IL22, and CXCL10 were significantly downregulated (q-value < 0.05). Transcription factor motif analysis at the differentially open chromatin regions, after anti-TNF treatment, revealed 58 transcription factor motifs enriched at the IL10 locus. We identified seven transcription factor candidates for the anti-TNF mediated regulation of IL-10, which were either differentially expressed or whose locus was differentially accessible upon anti-TNF treatment. Correlation analysis between the expression of these transcription factors and IL10 suggests a role for MAF, PRDM1, and/or EOMES in regulating IL10 expression in CD4+ T cells upon anti-TNF treatment.

IL-2 regulates expression of C-MAF in human CD4 T cells.

Blockade of IL-2R with humanized anti-CD25 Abs, such as daclizumab, inhibits Th2 responses in human T cells. Recent murine studies have shown that IL-2 also plays a significant role in regulating Th2 cell differentiation by activated STAT5. To explore the role of activated STAT5 in the Th2 differentiation of primary human T cells, we studied the mechanisms underlying IL-2 regulation of C-MAF expression. Chromatin immunoprecipitation studies revealed that IL-2 induced STAT5 binding to specific sites in the C-MAF promoter. These sites corresponded to regions enriched for markers of chromatin architectural features in both resting CD4 and differentiated Th2 cells. Unlike IL-6, IL-2 induced C-MAF expression in CD4 T cells with or without prior TCR stimulation. TCR-induced C-MAF expression was significantly inhibited by treatment with daclizumab or a JAK3 inhibitor, R333. Furthermore, IL-2 and IL-6 synergistically induced C-MAF expression in TCR-activated T cells, suggesting functional cooperation between these cytokines. Finally, both TCR-induced early IL4 mRNA expression and IL-4 cytokine expression in differentiated Th2 cells were significantly inhibited by IL-2R blockade. Thus, our findings demonstrate the importance of IL-2 in Th2 differentiation in human T cells and support the notion that IL-2R-directed therapies may have utility in the treatment of allergic disorders.

Multi-omics and machine learning reveal context-specific gene regulatory activities of PML::RARA in acute promyelocytic leukemia.

The PML::RARA fusion protein is the hallmark driver of Acute Promyelocytic Leukemia (APL) and disrupts retinoic acid signaling, leading to wide-scale gene expression changes and uncontrolled proliferation of myeloid precursor cells. While known to be recruited to binding sites across the genome, its impact on gene regulation and expression is under-explored. Using integrated multi-omics datasets, we characterize the influence of PML::RARA binding on gene expression and regulation in an inducible PML::RARA cell line model and APL patient ex vivo samples. We find that genes whose regulatory elements recruit PML::RARA are not uniformly transcriptionally repressed, as commonly suggested, but also may be upregulated or remain unchanged. We develop a computational machine learning implementation called Regulatory Element Behavior Extraction Learning to deconvolute the complex, local transcription factor binding site environment at PML::RARA bound positions to reveal distinct signatures that modulate how PML::RARA directs the transcriptional response.

MicroRNAs in lung diseases.

The advent of RNA sequencing technology has stimulated rapid advances in our understanding of the transcriptome, including discovery of the vast RNA complement generated by transcript splice variation and the expansion of our knowledge of non-coding RNAs. One non-coding RNA subtype, microRNAs (miRNAs), are particularly well studied, primarily because of their important roles as post-transcriptional gene regulators. The first miRNA was identified in the early 1990s and there are now thought to be around 1000 distinct miRNAs in man, with each cell type expressing a distinct repertoire. Increasing evidence has implicated miRNAs as having causative roles in a variety of lung diseases and has driven investigations into their potential as therapeutic targets.

Coordinated regulation of IL-4 and IL-13 expression in human T cells: 3C analysis for DNA looping.

Asthma is a chronic allergic disorder characterised by chronic inflammation. The balance of type I and type II (CD4+) T helper cells is of critical importance. In asthma there is an overexpression of T(H)2 cytokines, such as IL-4, IL-5 and IL-13. The genes encoding these cytokines are located together the same chromosomal region, 5q31.1 in humans. Here we confirm a central role for the transcription factors NFAT and GATA3 in the regulation of human IL-4 and IL-13. Chromatin Conformation Capture (3C) demonstrated the formation of specific ligation products containing spliced IL-4 and IL-13 DNA sequences in human T(H)2 polarised HuT-78 cells. This suggests that co-ordinate expression of T(H)2 cytokines, under the control of GATA3 and NFAT1 is due to the formation of a chromatin hub by DNA looping.

Diminished sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) expression contributes to airway remodelling in bronchial asthma.

Phenotypic modulation of airway smooth muscle (ASM) is an important feature of airway remodeling in asthma that is characterized by enhanced proliferation and secretion of pro-inflammatory chemokines. These activities are regulated by the concentration of free Ca(2+) in the cytosol ([Ca(2+)](i)). A rise in [Ca(2+)](i) is normalized by rapid reuptake of Ca(2+) into sarcoplasmic reticulum (SR) stores by the sarco/endoplasmic reticulum Ca(2+) (SERCA) pump. We examined whether increased proliferative and secretory responses of ASM from asthmatics result from reduced SERCA expression. ASM cells were cultured from subjects with and without asthma. SERCA expression was evaluated by western blot, immunohistochemistry and real-time PCR. Changes in [Ca(2+)](i), cell spreading, cellular proliferation, and eotaxin-1 release were measured. Compared with control cells from healthy subjects, SERCA2 mRNA and protein expression was reduced in ASM cells from subjects with moderately severe asthma. SERCA2 expression was similarly reduced in ASM in vivo in subjects with moderate/severe asthma. Rises in [Ca(2+)](i) following cell surface receptor-induced SR activation, or inhibition of SERCA-mediated Ca(2+) re-uptake, were attenuated in ASM cells from asthmatics. Likewise, the return to baseline of [Ca](i) after stimulation by bradykinin was delayed by approximately 50% in ASM cells from asthmatics. siRNA-mediated knockdown of SERCA2 in ASM from healthy subjects increased cell spreading, eotaxin-1 release and proliferation. Our findings implicate a deficiency in SERCA2 in ASM in asthma that contributes to its secretory and hyperproliferative phenotype in asthma, and which may play a key role in mechanisms of airway remodeling.