Trenton R. Schoeb

Transforming growth factor-beta induces development of the T(H)17 lineage.

A new lineage of effector CD4+ T cells characterized by production of interleukin (IL)-17, the T-helper-17 (TH17) lineage, was recently described based on developmental and functional features distinct from those of classical TH1 and TH2 lineages. Like TH1 and TH2, TH17 cells almost certainly evolved to provide adaptive immunity tailored to specific classes of pathogens, such as extracellular bacteria. Aberrant TH17 responses have been implicated in a growing list of autoimmune disorders. TH17 development has been linked to IL-23, an IL-12 cytokine family member that shares with IL-12 a common subunit, IL-12p40 (ref. 8). The IL-23 and IL-12 receptors also share a subunit, IL-12Rβ1, that pairs with unique, inducible components, IL-23R and IL-12Rβ2, to confer receptor responsiveness. Here we identify transforming growth factor-β (TGF-β) as a cytokine critical for commitment to TH17 development. TGF-β acts to upregulate IL-23R expression, thereby conferring responsiveness to IL-23. Although dispensable for the development of IL-17-producing T cells in vitro and in vivo, IL-23 is required for host protection against a bacterial pathogen, Citrobacter rodentium. The action of TGF-β on naive T cells is antagonized by interferon-γ and IL-4, thus providing a mechanism for divergence of the TH1, TH2 and TH17 lineages.

Transforming growth factor-β induces development of the TH17 lineage

A new lineage of effector CD4+ T cells characterized by production of interleukin (IL)-17, the T-helper-17 (TH17) lineage, was recently described based on developmental and functional features distinct from those of classical TH1 and TH2 lineages. Like TH1 and TH2, TH17 cells almost certainly evolved to provide adaptive immunity tailored to specific classes of pathogens, such as extracellular bacteria. Aberrant TH17 responses have been implicated in a growing list of autoimmune disorders. TH17 development has been linked to IL-23, an IL-12 cytokine family member that shares with IL-12 a common subunit, IL-12p40 (ref. 8). The IL-23 and IL-12 receptors also share a subunit, IL-12Rβ1, that pairs with unique, inducible components, IL-23R and IL-12Rβ2, to confer receptor responsiveness. Here we identify transforming growth factor-β (TGF-β) as a cytokine critical for commitment to TH17 development. TGF-β acts to upregulate IL-23R expression, thereby conferring responsiveness to IL-23. Although dispensable for the development of IL-17-producing T cells in vitro and in vivo, IL-23 is required for host protection against a bacterial pathogen, Citrobacter rodentium. The action of TGF-β on naive T cells is antagonized by interferon-γ and IL-4, thus providing a mechanism for divergence of the TH1, TH2 and TH17 lineages.

Requisite Role for the Dectin-1 β-Glucan Receptor in Pulmonary Defense against Aspergillus fumigatus

Immune suppression increases the incidence of invasive fungal infections, particularly those caused by the opportunistic mold Aspergillus fumigatus. Previous investigations revealed that members of the TLR family are not absolutely required for host defense against A. fumigatus in nonimmunosuppressed hosts, suggesting that other pattern recognition receptors are involved. We show in this study that naive mice (i.e., not pharmacologically immunosuppressed) lacking the β-glucan receptor Dectin-1 (Dectin-1−/−) are more sensitive to intratracheal challenge with A. fumigatus than control mice, exhibiting >80% mortality within 5 days, ultimately attributed to a compromise in respiratory mechanics. In response to A. fumigatus challenge, Dectin-1−/− mice demonstrated impaired IL-1α, IL-1β, TNF-α, CCL3/MIP-1α, CCL4/MIP-1β, and CXCL1/KC production, which resulted in insufficient lung neutrophil recruitment and uncontrolled A. fumigatus lung growth. Alveolar macrophages from Dectin-1−/− mice failed to produce proinflammatory mediators in response to A. fumigatus, whereas neutrophils from Dectin-1−/− mice had impaired reactive oxygen species production and impaired killing of A. fumigatus. We further show that IL-17 production in the lung after A. fumigatus challenge was Dectin-1 dependent, and that neutralization of IL-17 significantly impaired A. fumigatus clearance. Collectively, these results support a requisite role for Dectin-1 in in vivo defense against A. fumigatus.

A dominant, coordinated T regulatory cell-IgA response to the intestinal microbiota

A T cell receptor transgenic mouse line reactive to a microbiota flagellin, CBir1, was used to define mechanisms of host microbiota homeostasis. Intestinal IgA, but not serum IgA, was found to block mucosal flagellin uptake and systemic T cell activation in mice. Depletion of CD4+CD25+ Tregs decreased IgA+ B cells, total IgA, and CBir1-specific IgA in gut within days. Repletion of T cell-deficient mice with either CD4+CD25+ or CD4+foxp3+ Tregs restored intestinal IgA to a much greater extent than their reciprocal CD4+ subsets, indicating that Tregs are the major helper cells for IgA responses to microbiota antigens such as flagellin. We propose that the major role of this coordinated Treg-IgA response is to maintain commensalism with the microbiota.

Polycistronic Lentiviral Vector for “Hit and Run” Reprogramming of Adult Skin Fibroblasts to Induced Pluripotent Stem Cells

We report the derivation of induced pluripotent stem (iPS) cells from adult skin fibroblasts using a single, polycistronic lentiviral vector encoding the reprogramming factors Oct4, Sox2, and Klf4. Porcine teschovirus‐1 2A sequences that trigger ribosome skipping were inserted between human cDNAs for these factors, and the polycistron was subcloned downstream of the elongation factor 1 alpha promoter in a self‐inactivating (SIN) lentiviral vector containing a loxP site in the truncated 3′ long terminal repeat (LTR). Adult skin fibroblasts from a humanized mouse model of sickle cell disease were transduced with this single lentiviral vector, and iPS cell colonies were picked within 30 days. These cells expressed endogenous Oct4, Sox2, Nanog, alkaline phosphatase, stage‐specific embryonic antigen‐1, and other markers of pluripotency. The iPS cells produced teratomas containing tissue derived from all three germ layers after injection into immunocompromised mice and formed high‐level chimeras after injection into murine blastocysts. iPS cell lines with as few as three lentiviral insertions were obtained. Expression of Cre recombinase in these iPS cells resulted in deletion of the lentiviral vector, and sequencing of insertion sites demonstrated that remnant 291‐bp SIN LTRs containing a single loxP site did not interrupt coding sequences, promoters, or known regulatory elements. These results suggest that a single, polycistronic “hit and run” vector can safely and effectively reprogram adult dermal fibroblasts into iPS cells. Stem Cells 2009;27:1042–1049

Loss of Fibroblast Thy-1 Expression Correlates with Lung Fibrogenesis

Fibroblasts consist of heterogeneous subpopulations that have distinct roles in fibrotic responses. Previously we reported enhanced proliferation in response to fibrogenic growth factors and selective activation of latent transforming growth factor (TGF)-beta in fibroblasts lacking cell surface expression of Thy-1 glycoprotein, suggesting that Thy-1 modulates the fibrogenic potential of fibroblasts. Here we report that compared to controls Thy-1-/- C57BL/6 mice displayed more severe histopathological lung fibrosis, greater accumulation of lung collagen, and increased TGF-beta activation in the lungs 14 days after intratracheal bleomycin. The majority of cells demonstrating TGF-beta activation and myofibroblast differentiation in bleomycin-induced lesions were Thy-1-negative. Histological sections from patients with idiopathic pulmonary fibrosis demonstrated absent Thy-1 staining within fibroblastic foci. Normal lung fibroblasts, in both mice and humans, were predominantly Thy-1-positive. The fibrogenic cytokines interleukin-1 and tumor necrosis factor-alpha induced loss of fibroblast Thy-1 surface expression in vitro, which was associated with Thy-1 shedding, Smad phosphorylation, and myofibroblast differentiation. These results suggest that fibrogenic injury promotes loss of lung fibroblast Thy-1 expression, resulting in enhanced fibrogenesis.

Microbiota innate stimulation is a prerequisite for T cell spontaneous proliferation and induction of experimental colitis.

Little is known about how the microbiota regulates T cell proliferation and whether spontaneous T cell proliferation is involved in the pathogenesis of inflammatory bowel disease. In this study, we show that stimulation of innate pathways by microbiota-derived ligands and antigen-specific T cell stimulation are both required for intestinal inflammation. Microbiota-derived ligands promoted spontaneous T cell proliferation by activating dendritic cells (DCs) to produce IL-6 via Myd88, as shown by the spontaneous proliferation of T cells adoptively transferred into specific pathogen–free (SPF) RAG−/− mice, but not in germfree RAG−/− mice. Reconstitution of germfree RAG−/− mice with cecal bacterial lysate–pulsed DCs, but not with IL-6−/− or Myd88−/− DCs, restored spontaneous T cell proliferation. CBir1 TCR transgenic (CBir1 Tg) T cells, which are specific for an immunodominant microbiota antigen, induced colitis in SPF RAG−/− mice. Blocking the spontaneous proliferation of CBir1 Tg T cells by co-transferring bulk OT II CD4+ T cells abrogated colitis development. Although transferred OT II T cells underwent spontaneous proliferation in RAG−/− mice, the recipients failed to develop colitis because of the lack of cognate antigen in the intestinal lumen. Collectively, our data demonstrate that induction of colitis requires both spontaneous proliferation of T cells driven by microbiota-derived innate signals and antigen-specific T cell proliferation.

Transforming growth factor-β induces development of the T H 17 lineage

A new lineage of effector CD4+ T cells characterized by production of interleukin (IL)-17, the T-helper-17 (TH17) lineage, was recently described based on developmental and functional features distinct from those of classical TH1 and TH2 lineages. Like TH1 and TH2, TH17 cells almost certainly evolved to provide adaptive immunity tailored to specific classes of pathogens, such as extracellular bacteria. Aberrant TH17 responses have been implicated in a growing list of autoimmune disorders. TH17 development has been linked to IL-23, an IL-12 cytokine family member that shares with IL-12 a common subunit, IL-12p40 (ref. 8). The IL-23 and IL-12 receptors also share a subunit, IL-12Rβ1, that pairs with unique, inducible components, IL-23R and IL-12Rβ2, to confer receptor responsiveness. Here we identify transforming growth factor-β (TGF-β) as a cytokine critical for commitment to TH17 development. TGF-β acts to upregulate IL-23R expression, thereby conferring responsiveness to IL-23. Although dispensable for the development of IL-17-producing T cells in vitro and in vivo, IL-23 is required for host protection against a bacterial pathogen, Citrobacter rodentium. The action of TGF-β on naive T cells is antagonized by interferon-γ and IL-4, thus providing a mechanism for divergence of the TH1, TH2 and TH17 lineages.

Inactivation of Smad4 Accelerates KrasG12D-Mediated Pancreatic Neoplasia

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal human malignancies, with an overall 5-year survival rate of <5%. Genetic analysis of PDAC patient samples has shown that specific disease-associated mutations are correlated with histologically defined stages of neoplastic progression in the ductal epithelium. Activating mutations in KRAS are almost uniformly present in early-stage disease, with subsequent inactivating mutations in p16(INK4A), p53, and SMAD4 occurring in more advanced lesions. In this study, we have tested whether the loss of Smad4 would cooperate with an activating Kras(G12D) mutation to promote progression to PDAC using the Pdx1-Cre transgenic system to activate Kras(G12D) and delete Smad4 in all pancreatic lineages including the ductal epithelium. Analysis of double-mutant mice showed that loss of Smad4 significantly accelerated the progression of pancreatic intraepithelial neoplasias (mPanIN) and promoted a high incidence of intraductal papillary mucinous neoplasia and active fibrosis compared with Pdx1-Cre;Kras(G12D) or Pdx1-Cre;Smad4(lox/lox) mice. Occasionally, double-mutant mice progressed to locally invasive PDAC with little evidence of metastases by 6 months of age and without the detectable loss of p53 or p16(Ink4A) expression or function. The loss of Smad4 only seemed to promote disease progression in the presence of the activated Kras(G12D) allele because we observed no abnormal pathology within the pancreata of 23 Pdx1-Cre;Smad4(lox/lox) animals that were analyzed up to 8 months of age. This indicates that Smad4 is dispensable for normal pancreatic development but is critical for at least partial suppression of multiple Kras(G12D)-dependent disease-associated phenotypes.

Intercellular adhesion molecule-1 expression is required on multiple cell types for the development of experimental autoimmune encephalomyelitis.

Many members of the Ig superfamily of adhesion molecules, such as ICAM-1 and VCAM-1, have been implicated in the pathogenesis of multiple sclerosis. Although it is well-established that VCAM-1/VLA-4 interactions can play important roles in mediating CNS inflammatory events in multiple sclerosis patients and during the development of experimental allergic encephalomyelitis (EAE), the contributions of ICAM-1 are poorly understood. This is due in large part to conflicting results from Ab inhibition studies and the observation of exacerbated EAE in ICAM-1 mutant mice that express a restricted set of ICAM-1 isoforms. To determine ICAM-1-mediated mechanisms in EAE, we analyzed ICAM-1 null mutant mice (ICAM-1null), which express no ICAM-1 isoforms. ICAM-1null mice had significantly attenuated EAE characterized by markedly reduced spinal cord T cell infiltration and IFN-γ production by these cells. Adoptive transfer of Ag-restimulated T cells from wild-type to ICAM-1null mice or transfer of ICAM-1null Ag-restimulated T cells to control mice failed to induce EAE. ICAM-1null T cells also showed reduced proliferative capacity and substantially reduced levels of IFN-γ, TNF-α, IL-4, IL-10, and IL-12 compared with that of control T cells following myelin oligodendrocyte glycoprotein 35–55 restimulation in vitro. Our results indicate that ICAM-1 expression is critical on T cells and other cell types for the development of demyelinating disease and suggest that expression of VCAM-1 and other adhesion molecules cannot fully compensate for the loss of ICAM-1 during EAE development.