Droplet-based forward genetic screening of astrocyte-microglia cross-talk.

Cell-cell interactions in the central nervous system play important roles in neurologic diseases. However, little is known about the specific molecular pathways involved, and methods for their systematic identification are limited. Here, we developed a forward genetic screening platform that combines CRISPR-Cas9 perturbations, cell coculture in picoliter droplets, and microfluidic-based fluorescence-activated droplet sorting to identify mechanisms of cell-cell communication. We used SPEAC-seq (systematic perturbation of encapsulated associated cells followed by sequencing), in combination with in vivo genetic perturbations, to identify microglia-produced amphiregulin as a suppressor of disease-promoting astrocyte responses in multiple sclerosis preclinical models and clinical samples. Thus, SPEAC-seq enables the high-throughput systematic identification of cell-cell communication mechanisms.

Antigen-Specific Treg Therapy in Type 1 Diabetes - Challenges and Opportunities.

Regulatory T cells (Tregs) are key mediators of peripheral self-tolerance and alterations in their frequencies, stability, and function have been linked to autoimmunity. The antigen-specific induction of Tregs is a long-envisioned goal for the treatment of autoimmune diseases given reduced side effects compared to general immunosuppressive therapies. However, the translation of antigen-specific Treg inducing therapies for the treatment or prevention of autoimmune diseases into the clinic remains challenging. In this mini review, we will discuss promising results for antigen-specific Treg therapies in allergy and specific challenges for such therapies in autoimmune diseases, with a focus on type 1 diabetes (T1D). We will furthermore discuss opportunities for antigen-specific Treg therapies in T1D, including combinatorial strategies and tissue-specific Treg targeting. Specifically, we will highlight recent advances in miRNA-targeting as a means to foster Tregs in autoimmunity. Additionally, we will discuss advances and perspectives of computational strategies for the detailed analysis of tissue-specific Tregs on the single-cell level.

Advances in Human Immune System Mouse Models for Personalized Treg-Based Immunotherapies.

Immunodeficient mice engrafted with a functional human immune system [Human immune system (HIS) mice] have paved the way to major advances for personalized medicine and translation of immune-based therapies. One prerequisite for advancing personalized medicine is modeling the immune system of individuals or disease groups in a preclinical setting. HIS mice engrafted with peripheral blood mononuclear cells have provided fundamental insights in underlying mechanisms guiding immune activation vs. regulation in several diseases including cancer. However, the development of Graft-vs.-host disease restrains relevant long-term studies in HIS mice. Alternatively, engraftment with hematopoietic stem cells (HSCs) enables mimicking different disease stages, however, low frequencies of HSCs in peripheral blood of adults impede engraftment efficacy. One possibility to overcome those limitations is the use of patient-derived induced pluripotent stem cells (iPSCs) reprogrammed into HSCs, a challenging process which has recently seen major advances. Personalized HIS mice bridge research in mice and human diseases thereby facilitating the translation of immunomodulatory therapies. Regulatory T cells (Tregs) are important mediators of immune suppression and thereby contribute to tumor immune evasion, which has made them a central target for cancer immunotherapies. Importantly, studying Tregs in the human immune system in vivo in HIS mice will help to determine requirements for efficient Treg-targeting. In this review article, we discuss advances on personalized HIS models using reprogrammed iPSCs and review the use of HIS mice to study requirements for efficient targeting of human Tregs for personalized cancer immunotherapies.

Aging Aggravates Cachexia in Tumor-Bearing Mice.

BACKGROUND: Cancer is primarily a disease of high age in humans, yet most mouse studies on cancer cachexia are conducted using young adolescent mice. Given that metabolism and muscle function change with age, we hypothesized that aging may affect cachexia progression in mouse models. METHODS: We compare tumor and cachexia development in young and old mice of three different strains (C57BL/6J, C57BL/6N, BALB/c) and with two different tumor cell lines (Lewis Lung Cancer, Colon26). Tumor size, body and organ weights, fiber cross-sectional area, circulating cachexia biomarkers, and molecular markers of muscle atrophy and adipose tissue wasting are shown. We correlate inflammatory markers and body weight dependent on age in patients with cancer. RESULTS: We note fundamental differences between mouse strains. Aging aggravates weight loss in LLC-injected C57BL/6J mice, drives it in C57BL/6N mice, and does not influence weight loss in C26-injected BALB/c mice. Glucose tolerance is unchanged in cachectic young and old mice. The stress marker GDF15 is elevated in cachectic BALB/c mice independent of age and increased in old C57BL/6N and J mice. Inflammatory markers correlate significantly with weight loss only in young mice and patients. CONCLUSIONS: Aging affects cachexia development and progression in mice in a strain-dependent manner and influences the inflammatory profile in both mice and patients. Age is an important factor to consider for future cachexia studies.

miRNA142-3p targets Tet2 and impairs Treg differentiation and stability in models of type 1 diabetes.

In type 1 diabetes, the appearance of islet autoantibodies indicates the onset of islet autoimmunity, often many years before clinical symptoms arise. While T cells play a major role in the destruction of pancreatic beta cells, molecular underpinnings promoting aberrant T cell activation remain poorly understood. Here, we show that during islet autoimmunity an miR142-3p/Tet2/Foxp3 axis interferes with the efficient induction of regulatory T (Treg) cells, resulting in impaired Treg stability in mouse and human. Specifically, we demonstrate that miR142-3p is induced in islet autoimmunity and that its inhibition enhances Treg induction and stability, leading to reduced islet autoimmunity in non-obese diabetic mice. Using various cellular and molecular approaches we identify Tet2 as a direct target of miR142-3p, thereby linking high miR142-3p levels to epigenetic remodeling in Tregs. These findings offer a mechanistic model where during islet autoimmunity miR142-3p/Tet2-mediated Treg instability contributes to autoimmune activation and progression.

Immunometabolism and atherosclerosis: perspectives and clinical significance: a position paper from the Working Group on Atherosclerosis and Vascular Biology of the European Society of Cardiology.

Inflammation is an important driver of atherosclerosis, and the favourable outcomes of the Canakinumab Anti-inflammatory Thrombosis Outcome Study (CANTOS) trial revealed the large potential of anti-inflammatory drugs for the treatment of cardiovascular disease, especially in patients with a pro-inflammatory constitution. However, the complex immune reactions driving inflammation in the vascular wall in response to an atherosclerotic microenvironment are still being unravelled. Novel insights into the cellular processes driving immunity and inflammation revealed that alterations in intracellular metabolic pathways are strong drivers of survival, growth, and function of immune cells. Therefore, this position paper presents a brief overview of the recent developments in the immunometabolism field, focusing on its role in atherosclerosis. We will also highlight the potential impact of immunometabolic markers and targets in clinical cardiovascular medicine.

A Stat6/Pten Axis Links Regulatory T Cells with Adipose Tissue Function.

Obesity and type 2 diabetes are associated with metabolic defects and adipose tissue inflammation. Foxp3+ regulatory T cells (Tregs) control tissue homeostasis by counteracting local inflammation. However, if and how T cells interlink environmental influences with adipocyte function remains unknown. Here, we report that enhancing sympathetic tone by cold exposure, beta3-adrenergic receptor (ADRB3) stimulation or a short-term high-calorie diet enhances Treg induction in vitro and in vivo. CD4+ T cell proteomes revealed higher expression of Foxp3 regulatory networks in response to cold or ADRB3 stimulation in vivo reflecting Treg induction. Specifically, Ragulator-interacting protein C17orf59, which limits mTORC1 activity, was upregulated in CD4+ T cells by either ADRB3 stimulation or cold exposure, suggesting contribution to Treg induction. By loss- and gain-of-function studies, including Treg depletion and transfers in vivo, we demonstrated that a T cell-specific Stat6/Pten axis links cold exposure or ADRB3 stimulation with Foxp3+ Treg induction and adipose tissue function. Our findings offer a new mechanistic model in which tissue-specific Tregs maintain adipose tissue function.

miRNA92a targets KLF2 and the phosphatase PTEN signaling to promote human T follicular helper precursors in T1D islet autoimmunity.

Aberrant immune activation mediated by T effector cell populations is pivotal in the onset of autoimmunity in type 1 diabetes (T1D). T follicular helper (TFH) cells are essential in the induction of high-affinity antibodies, and their precursor memory compartment circulates in the blood. The role of TFH precursors in the onset of islet autoimmunity and signaling pathways regulating their differentiation is incompletely understood. Here, we provide direct evidence that during onset of islet autoimmunity, the insulin-specific target T-cell population is enriched with a C-X-C chemokine receptor type 5 (CXCR5)+CD4+ TFH precursor phenotype. During onset of islet autoimmunity, the frequency of TFH precursors was controlled by high expression of microRNA92a (miRNA92a). miRNA92a-mediated TFH precursor induction was regulated by phosphatase and tension homolog (PTEN) - phosphoinositol-3-kinase (PI3K) signaling involving PTEN and forkhead box protein O1 (Foxo1), supporting autoantibody generation and triggering the onset of islet autoimmunity. Moreover, we identify Krueppel-like factor 2 (KLF2) as a target of miRNA92a in regulating human TFH precursor induction. Importantly, a miRNA92a antagomir completely blocked induction of human TFH precursors in vitro. More importantly, in vivo application of a miRNA92a antagomir to nonobese diabetic (NOD) mice with ongoing islet autoimmunity resulted in a significant reduction of TFH precursors in peripheral blood and pancreatic lymph nodes. Moreover, miRNA92a antagomir application reduced immune infiltration and activation in pancreata of NOD mice as well as humanized NOD Scid IL2 receptor gamma chain knockout (NSG) human leucocyte antigen (HLA)-DQ8 transgenic animals. We therefore propose that miRNA92a and the PTEN-PI3K-KLF2 signaling network could function as targets for innovative precision medicines to reduce T1D islet autoimmunity.

Follicular Helper T Cells in Autoimmunity.

The development of multiple disease-relevant autoantibodies is a hallmark of autoimmune diseases. In autoimmune type 1 diabetes (T1D), a variable time frame of autoimmunity precedes the clinically overt disease. The relevance of T follicular helper (TFH) cells for the immune system is increasingly recognized. Their pivotal contribution to antibody production by providing help to germinal center (GC) B cells facilitates the development of a long-lived humoral immunity. Their complex differentiation process, involving various stages and factors like B cell lymphoma 6 (Bcl6), is strictly controlled, as anomalous regulation of TFH cells is connected with immunopathologies. While the adverse effects of a TFH cell-related insufficient humoral immunity are obvious, the role of increased TFH frequencies in autoimmune diseases like T1D is currently highlighted. High levels of autoantigen trigger an excessive induction of TFH cells, consequently resulting in the production of autoantibodies. Therefore, TFH cells might provide promising approaches for novel therapeutic strategies.

Type 1 diabetes vaccine candidates promote human Foxp3(+)Treg induction in humanized mice.

Immune tolerance is executed partly by Foxp3(+)regulatory T (Treg) cells, which suppress autoreactive T cells. In autoimmune type 1 diabetes (T1D) impaired tolerance promotes destruction of insulin-producing ß-cells. The development of autoantigen-specific vaccination strategies for Foxp3(+)Treg-induction and prevention of islet autoimmunity in patients is still in its infancy. Here, using human haematopoietic stem cell-engrafted NSG-HLA-DQ8 transgenic mice, we provide direct evidence for human autoantigen-specific Foxp3(+)Treg-induction in vivo. We identify HLA-DQ8-restricted insulin-specific CD4(+)T cells and demonstrate efficient human insulin-specific Foxp3(+)Treg-induction upon subimmunogenic vaccination with strong agonistic insulin mimetopes in vivo. Induced human Tregs are stable, show increased expression of Treg signature genes such as Foxp3, CTLA4, IL-2Rα and TIGIT and can efficiently suppress effector T cells. Such Foxp3(+)Treg-induction does not trigger any effector T cells. These T1D vaccine candidates could therefore represent an expedient improvement in the challenge to induce human Foxp3(+)Tregs and to develop novel precision medicines for prevention of islet autoimmunity in children at risk of T1D.

Cyclosporine A regulates pro-inflammatory cytokine production in ulcerative colitis.

Crohn's disease (CD) and ulcerative colitis (UC) are the two major forms of inflammatory bowel diseases (IBD), which are defined as relapsing inflammations of the gastrointestinal tract. Cyclosporine A (CsA) is a potential rescue treatment to avoid colectomy in severe steroid-refractory UC patients. The molecular mechanism of action of CsA in UC is nevertheless still not well understood. The aim of this study was to investigate the effect of CsA on a possible modulation of cytokine production by peripheral blood mononuclear cells (PBMCs) of controls and patients with UC or CD. Upon CsA treatment, analyses of cytokine levels revealed a significant reduction of IL-13 expression in PBMCs from patients with UC, whereas other cytokine expression levels remained unaffected. To address the question whether CsA treatment impinges on the induction of cell death, apoptosis assays were performed using CD4(+) T cells from peripheral blood of patients suffering from either UC or CD. It became clear that CsA treatment resulted in a specific induction of apoptosis in samples from controls and patients with UC but not with CD. Apoptosis induction was not mediated via the mitochondrial apoptosis pathway. The present data support the concept that CsA treatment modulates pro-inflammatory cytokine production and T cell survival in UC via the induction of apoptosis and might therefore help to explain the clinical efficacy of CsA in patients with UC.

Nuclear factor of activated T cells - a transcription factor family as critical regulator in lung and colon cancer.

Nuclear factor of activated T cells (NFAT) was first identified as a transcription factor which is activated upon T cell stimulation. Subsequent studies uncovered that a whole family of individual NFAT proteins exists with pleiotropic functions not only in immune but also in nonimmune cells. However, dysregulation of NFAT thereby favors malignant growth and cancer. Summarizing the recent advances in understanding how individual NFAT factors regulate the immune system, this review gives new insights into the critical role of NFAT in cancer development with special focus on inflammation-associated colorectal cancer.

Therapeutic opportunities for manipulating T(Reg) cells in autoimmunity and cancer.

Forkhead box P3 (FOXP3)-expressing regulatory T (T(Reg)) cells have a pivotal role in the regulation of immune responses and in the maintenance of immunological self-tolerance. These cells have emerged as attractive targets for strategies that allow the steering of immune responses in desired directions - arming the immune system to destroy infected cells and cancer cells or downregulating it to limit tissue destruction in autoimmunity. Efforts to understand the generation, activation and function of T(Reg) cells should permit the development of therapeutics for reprogramming the immune system. In this Review, we discuss insights into the generation of T(Reg) cells, their involvement in disease and the molecular basis of the dominant tolerance exerted by FOXP3(+) T(Reg) cells that could permit their safe and specific manipulation in humans.

Transcription factor NFATc2 controls the emergence of colon cancer associated with IL-6-dependent colitis.

NFAT transcription factors control T-cell activation and function. Specifically, the transcription factor NFATc2 affects the regulation of cell differentiation and growth and plays a critical role in the development of colonic inflammation. Here, we used an experimental model of colitis-associated colorectal carcinoma to investigate the contribution of NFATc2 to the promotion of colonic tumors. Compared with wild-type animals that readily presented with multiple colon tumors, NFATc2-deficient mice were protected from tumor development. This observed decrease in colonic tumor progression was associated with reduced endoscopic inflammation, increased apoptosis of lamina propria T lymphocytes, and significantly reduced levels of the critical proinflammatory cytokines interleukin (IL)-21 and IL-6. Administration of hyper IL-6 abrogated protection from tumor progression in NFATc2-knockout mice and restored tumor incidence to control levels. Taken together, our findings highlight a pivotal role for NFATc2 in the establishment of inflammation-associated colorectal tumors mediated by control of IL-6 expression.

Immune modulatory treatment of trinitrobenzene sulfonic acid colitis with calcitriol is associated with a change of a T helper (Th) 1/Th17 to a Th2 and regulatory T cell profile.

A number of recent studies testify that calcitriol alone or in combination with corticosteroids exerts strong immune modulatory activity. As a new approach, we evaluated the protolerogenic potential of calcitriol and dexamethasone in acute T helper (Th)1-mediated colitis in mice. A rectal enema of trinitrobenzene sulfonic acid (TNBS) (100 mg/kg) was applied to BALB/c mice. Calcitriol and/or dexamethasone were administered i.p. from days 0 to 3 or 3 to 5 following the instillation of the haptenating agent. Assessment of colitis severity was performed daily. Colon tissue was analyzed macroscopically and microscopically, and myeloperoxidase activity, as well as cytokine levels [tumor necrosis factor-alpha, interferon-gamma, interleukin (IL)-12p70, IL-1beta, IL-10, IL-4] were determined by enzyme-linked immunosorbent assay, T-bet, GATA family of transcription factors 3, a Th2 master regulator (GATA3), Foxp3, cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), IL-23p19 and IL-17 expression by immunoblot analysis. The combination of the steroids most effectively reduced the clinical and histopathologic severity of TNBS colitis. Th1-related parameters were down-regulated, whereas Th2 markers like IL-4 and GATA3 were up-regulated. Apart from known steroid effects, calcitriol in particular promoted regulatory T cell profiles as indicated by a marked increase of IL-10, TGFbeta, FoxP3, and CTLA4. Furthermore, analysis of dendritic cell mediators responsible for a proinflammatory differentiation of T cells revealed a significant reduction of IL-12p70 and IL23p19 as well as IL-6 and IL-17. Thus, our data support a rationale for a steroid-sparing, clinical application of calcitriol derivatives in inflammatory bowel disease. Furthermore they suggest that early markers of inflammatory dendritic cell and Th17 differentiation qualify as new target molecules for both calcitriol and highly selective immune-modulating vitamin D analogs.

The TGFbeta/Smad 3-signaling pathway is involved in butyrate-mediated vitamin D receptor (VDR)-expression.

Previously, we demonstrated the pivotal role of the vitamin D receptor (VDR) in mediating the butyrate-induced differentiation in colon cancer cells. Smad 3, a downstream component of transforming growth factor-beta (TGFbeta) signaling, has been shown to act as a coactivator of VDR and to possibly regulate the vitamin D signaling pathway. In this study, we demonstrate a distinct impact of the TGFbeta/Smad 3-signaling pathway in the butyrate-mediated VDR expression and induction of differentiation. Butyrate treatment resulted in a significant induction of the phosphorylation level of Smad 3, while the combination of butyrate and a specific TGFbeta1-antibody or a TGFbeta-receptor inhibitor considerably diminished the butyrate-induced upregulation of VDR expression. Using a specific inhibitor, we were also able to demonstrate an involvement of the p38 MAPK in the increase of Smad 3 phosphorylation following butyrate treatment, thus opening the view to further elucidate possible mechanisms mediating the upregulation of VDR expression following butyrate treatment in colon cancer cells.

Inhibition of breast cancer cell adhesion and bone metastasis by the extracellular adherence protein of Staphylococcus aureus.

Bone metastasis is a common sequelae of breast cancer and the interaction of alpha v beta3-integrin with osteopontin (OPN) found in the extracellular matrix of mineralized tissues is implicated in this process. The integrin-dependent proadhesive and promigratory functions of OPN are particularly attributed to the 40 kD N-terminal fragment that derives upon matrix metalloproteinase (MMP) cleavage. Based on the broad repertoire of interactions between Staphylococcus aureus extracellular adherence protein (Eap) and host components, we here characterized Eap to specifically interact with recombinant full-length OPN and the 40 kD N-terminal MMP cleavage fragment, but not with the 32 kD or the 25 kD C-terminal fragments of OPN. Eap thereby prevented the OPN/alpha v beta3-integrin interaction, as well as the alpha v beta3-integrin-dependent adhesion of MDA-MB-231 breast cancer cells to full-length OPN or to the 40 kD fragment and the migration of these cells towards OPN. Furthermore, Eap treatment markedly impaired the development of osseous metastasis of human MDA-MB-231 cells in vivo. Taken together, Eap may represent an attractive novel treatment for the prevention of breast cancer bone metastasis.

FTY720 ameliorates Th1-mediated colitis in mice by directly affecting the functional activity of CD4+CD25+ regulatory T cells.

Following the present concepts, the synthetic sphingosine analog of myriocin FTY720 alters migration and homing of lymphocytes via sphingosine 1-phosphate receptors. However, several studies indicate that the immunosuppressive properties of FTY720 may alternatively be due to tolerogenic activities via modulation of dendritic cell differentiation or based on direct effects on CD4(+)CD25(+) regulatory T cells (Treg). As Treg play an important role for the cure of inflammatory colitis, we used the Th1-mediated 2,4,6-trinitrobenzene sulfonic acid (TNBS) colitis model to address the therapeutic potential of FTY720 in vivo. A rectal enema of TNBS was given to BALB/c mice. FTY720 was administered i.p. from days 0 to 3 or 3 to 5. FTY720 substantially reduced all clinical, histopathologic, macroscopic, and microscopic parameters of colitis analyzed. The therapeutic effects of FTY720 were associated with a down-regulation of IL-12p70 and subsequent Th1 cytokines. Importantly, FTY720 treatment resulted in a prominent up-regulation of FoxP3, IL-10, TGFbeta, and CTLA4. Supporting the hypothesis that FTY720 directly affects functional activity of CD4(+)CD25(+) Treg, we measured a significant increase of CD25 and FoxP3 expression in isolated lamina propria CD4(+) T cells of FTY720-treated mice. The impact of FTY720 on Treg induction was further confirmed by concomitant in vivo blockade of CTLA4 or IL-10R which significantly abrogated its therapeutic activity. In conclusion, our data provide clear evidence that in addition to its well-established effects on migration FTY720 leads to a specific down-regulation of proinflammatory signals while simultaneously inducing functional activity of CD4(+)CD25(+) Treg. Thus, FTY720 may offer a promising new therapeutic strategy for the treatment of IBD.

The new low calcemic vitamin D analog 22-ene-25-oxa-vitamin D prominently ameliorates T helper cell type 1-mediated colitis in mice.

In addition to its well defined role as a key regulator of calcium and bone metabolism, 1,25-dihydroxyvitamin D(3) (calcitriol) has been established as a potent modulator of immune cell function. Still, because of the hypercalcemic toxicity occurring after systemic application of the parent compound, its clinical application as an immunosuppressant has been hampered. Recently, we described 22-ene-25-oxa-vitamin D (ZK156979) as a representative of a novel class of low calcemic vitamin D analogs with well preserved immunosuppressive activity in vitro. Here, in vivo colitis was induced by applying a rectal enema of 2,4,6-trinitrobenzene sulfonic acid (TNBS) to male BALB/c mice, and calcitriol (0.2 microg/kg) or ZK156979 (0.1-2.0 microg/kg) was given i.p. from days 0 to 3 or 3 to 5. Body mass and clinical activity score of colitis were recorded daily. Colon tissue was analyzed macroscopically and microscopically, myeloperoxidase activity and cytokine levels [tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, interleukin (IL)-10, and IL-4] were determined by enzyme-linked immunosorbent assay, and T-box transcription factor (T-bet) expression was determined by immunoblot analysis. We found that treatment with ZK156979 clearly reduced the severity of TNBS-induced colitis without exhibiting calcemic effects. Both early and late treatment abrogated body weight loss, diarrhea, and macroscopic intestinal inflammation with a potency comparable with that of calcitriol. The therapeutic effect of ZK156979 was accompanied by a down-regulation of myeloperoxidase activity, TNF-alpha, IFN-gamma, and T-bet expression decreased, whereas local tissue IL-10 and IL-4 protein levels increased. To conclude, our data provide the first clear evidence that ZK156979 exhibits a beneficial prophylactic as well as therapeutic profile in T helper cell type 1-like experimental colitis, offering new therapeutic options for the treatment of human inflammatory bowel diseases.

Upregulation of 25-hydroxyvitamin D(3)-1(alpha)-hydroxylase by butyrate in Caco-2 cells.

AIM: To investigate the possible involvement of 25-hydroxyvitamin D(3)-1(alpha)-hydroxylase [1alpha-25(OH) (2) D(3)] in butyrate-induced differentiation in human intestinal cell line Caco-2 cells. METHODS: Caco-2 cells were incubated either with 3 mmol/L butyrate and 1 micromol/L 25(OH) (2) D(3) or with 1 micromol/L 1alpha-25(OH) (2) D(3) for various time intervals ranging from 0 to 72 h. Additionally, cells were co-incubated with butyrate and either 25(OH) (2) D(3) or 1alpha-25(OH) (2) D(3). 1alpha-25(OH) (2) D(3) mRNA was determined semi-quantitatively using the fluorescent dye PicoGreen. Immunoblotting was used for the detection of 1alpha-25(OH) (2) D(3) protein. Finally, enzymatic activity was measured by ELISA. RESULTS: Both butyrate and 1alpha-25(OH) (2) D(3) stimulated differentiation of Caco-2 cells after a 48 h incubation period, while 25(OH) (2) D(3) had no impact on cell differentiation. Synergistic effects on differentiation were observed when cells were co-incubated with butyrate and vitamin D metabolite. Butyrate transiently upregulated 1alpha-25(OH) (2) D(3) mRNA followed by a timely delayed protein upregulation. Coincidently, enzymatic activity was enhanced significantly. The induction of the enzyme allowed for comparable differentiating effects of both vitamin D metabolites. CONCLUSION: Our experimental data provide a further mechanism for the involvement of the vitamin D signaling pathway in colonic epithelial cell differentiation by butyrate. The enhancement of 1alpha-25(OH) (2) D(3) followed by antiproliferative effects of the vitamin D prohormone in the Caco-2 cell line suggest that 25(OH) (2) D(3) in combination with butyrate may offer a new therapeutic approach for the treatment of colon cancer.