Hedgehog signaling in the airway epithelium of patients with chronic obstructive pulmonary disease.

Genome-wide association studies have linked gene variants of the receptor patched homolog 1 (PTCH1) with chronic obstructive pulmonary disease (COPD). However, its biological role in the disease is unclear. Our objective was to determine the expression pattern and biological role of PTCH1 in the lungs of patients with COPD. Airway epithelial-specific PTCH1 protein expression and epithelial morphology were assessed in lung tissues of control and COPD patients. PTCH1 mRNA expression was measured in bronchial epithelial cells obtained from individuals with and without COPD. The effects of PTCH1 siRNA knockdown on epithelial repair and mucous expression were evaluated using human epithelial cell lines. Ptch1+/- mice were used to assess the effect of decreased PTCH1 on mucous expression and airway epithelial phenotypes. Airway epithelial-specific PTCH1 protein expression was significantly increased in subjects with COPD compared to controls, and its expression was associated with total airway epithelial cell count and thickness. PTCH1 knockdown attenuated wound closure and mucous expression in airway epithelial cell lines. Ptch1+/- mice had reduced mucous expression compared to wildtype mice following mucous induction. PTCH1 protein is up-regulated in COPD airway epithelium and may upregulate mucous expression. PTCH1 provides a novel target to reduce chronic bronchitis in COPD patients.

Microbiome-driven allergic lung inflammation is ameliorated by short-chain fatty acids.

The mammalian gastrointestinal tract harbors a microbial community with metabolic activity critical for host health, including metabolites that can modulate effector functions of immune cells. Mice treated with vancomycin have an altered microbiome and metabolite profile, exhibit exacerbated T helper type 2 cell (Th2) responses, and are more susceptible to allergic lung inflammation. Here we show that dietary supplementation with short-chain fatty acids (SCFAs) ameliorates this enhanced asthma susceptibility by modulating the activity of T cells and dendritic cells (DCs). Dysbiotic mice treated with SCFAs have fewer interleukin-4 (IL4)-producing CD4+ T cells and decreased levels of circulating immunoglobulin E (IgE). In addition, DCs exposed to SCFAs activate T cells less robustly, are less motile in response to CCL19 in vitro, and exhibit a dampened ability to transport inhaled allergens to lung draining nodes. Our data thus demonstrate that gut dysbiosis can exacerbate allergic lung inflammation through both T cell- and DC-dependent mechanisms that are inhibited by SCFAs.

Mucosal production of uric acid by airway epithelial cells contributes to particulate matter-induced allergic sensitization.

Exposure to particulate matter (PM), a major component of air pollution, contributes to increased morbidity and mortality worldwide. PM induces innate immune responses and contributes to allergic sensitization, although the mechanisms governing this process remain unclear. Lung mucosal uric acid has also been linked to allergic sensitization. The links among PM exposure, uric acid, and allergic sensitization remain unexplored. We therefore investigated the mechanisms behind PM-induced allergic sensitization in the context of lung mucosal uric acid. PM10 and house dust mite exposure selectively induced lung mucosal uric acid production and secretion in vivo, which did not occur with other challenges (lipopolysaccharide, virus, bacteria, or inflammatory/fibrotic stimuli). PM10-induced uric acid mediates allergic sensitization and augments antigen-specific T-cell proliferation, which is inhibited by uricase. We then demonstrate that human airway epithelial cells secrete uric acid basally and after stimulation through a previously unidentified mucosal secretion system. Our work discovers a previously unknown mechanism of air pollution-induced, uric acid-mediated, allergic sensitization that may be important in the pathogenesis of asthma.

In situ hematopoiesis: a regulator of TH2 cytokine-mediated immunity and inflammation at mucosal surfaces.

Hematopoiesis refers to the development of blood cells in the body through the differentiation of pluripotent stem cells. Although hematopoiesis is a multifocal process during embryonic development, under homeostatic conditions it occurs exclusively within the bone marrow. There, a limited number of hematopoietic stem cells differentiate into a rapidly proliferating population of lineage-restricted progenitors that serve to replenish circulating blood cells. However, emerging reports now suggest that under inflammatory conditions, alterations in hematopoiesis that occur outside of the bone marrow appear to constitute a conserved mechanism of innate immunity. Moreover, recent reports have identified previously unappreciated pathways that regulate the egress of hematopoietic progenitor cells from the bone marrow, alter their activation status, and skew their developmental potential. These studies suggest that progenitor cells contribute to inflammatory response by undergoing in situ hematopoiesis (ISH). In this review, we highlight the differences between homeostatic hematopoiesis, which occurs in the bone marrow, and ISH, which occurs at mucosal surfaces. Further, we highlight factors produced at local sites of inflammation that regulate hematopoietic progenitor cell responses and the development of TH2 cytokine-mediated inflammation. Finally, we discuss the therapeutic potential of targeting ISH in preventing the development of inflammation at mucosal sites.

Myeloid cell-specific expression of Ship1 regulates IL-12 production and immunity to helminth infection.

Helminth infection leads to the local proliferation and accumulation of macrophages in tissues. However, the function of macrophages during helminth infection remains unclear. SH2-containing inositol 5'-phosphatase 1 (Ship1, Inpp5d) is a lipid phosphatase that has been shown to play a critical role in macrophage function. Here, we identify a critical role for Ship1 in the negative regulation of interleukin (IL)-12/23p40 production by macrophages during infection with the intestinal helminth parasite Trichuris muris. Mice with myeloid cell-specific deletion of Ship1 (Ship1(ΔLysM) mice) develop a non-protective T-helper type 1 cell response and fail to expel parasites. Ship1-deficient macrophages produce heightened levels of IL-12/23p40 in vitro and in vivo and antibody blockade of IL-12/23p40 renders Ship1(ΔLysM) mice resistant to Trichuris infection. Our results identify a critical role for the negative regulation of IL-12/23p40 production by macrophages in the development of a protective T(H)2 cell response.

NUP98-HOXA10hd-expanded hematopoietic stem cells efficiently reconstitute bone marrow of mismatched recipients and induce tolerance.

Gene therapy as well as methods capable of returning cells to a pluripotent state (iPS) have enabled the correction of genetic deficiencies in syngenic adult progenitors, reducing the need for immunosuppression in cell therapy approaches. However, in diseases involving mutations that lead to the complete lack of a protein, such as Duchenne muscular dystrophy, the main immunogens leading to rejection of transplanted cells are the therapeutic proteins themselves. In these cases even iPS cells would not circumvent the need for immunosuppression, and alternative strategies must be developed. One such potential strategy seeks to induce immune tolerance using hematopoietic stem cells originated from the same donor or iPS line from which the therapeutic progenitors are derived. However, donor hematopoietic stem cells (HSCs) are available in limiting numbers and embryonic stem (ES) cell-derived HSCs engraft poorly in adults. While these limitations have been circumvented by ectopic expression of HOXB4, overexpression of this protein is associated with inefficient lymphoid reconstitution. Here we show that adult HSCs expanded with a NUP98- HOXA10hd fusion protein sustain long-term engraftment in immunologically mismatched recipients and generate normal numbers of lymphoid cells. In addition, NUP98-HOXA10hd-expanded cells induce functional immune tolerance to a subsequent transplant of myogenic progenitors immunologically matched with the transplanted HSCs.

The Rap GTPases regulate the migration, invasiveness and in vivo dissemination of B-cell lymphomas.

B-cell lymphomas are common malignancies in which transformed B cells enter the circulation, extravasate into tissues and form tumors in multiple organs. Lymphoma cells are thought to exit the vasculature and enter tissues through the same chemokine- and adhesion molecule-dependent mechanisms as normal B cells. We have previously shown that activation of the Rap GTPases, proteins that control cytoskeletal organization and integrin activation, is critical for chemokine-induced migration and adhesion in B-lymphoma cell lines. Using the A20 murine B-lymphoma cell line as a model, we now show that Rap activation is important for circulating lymphoma cells to enter tissues and form tumors in vivo. In vitro assays showed that Rap activation is required for A20 cells to efficiently adhere to vascular endothelial cells and undergo transendothelial migration. These findings suggest that Rap or its effectors could be novel targets for treating B-cell lymphomas.

Anuria, omphalocele, and perinatal lethality in mice lacking the CD34-related protein podocalyxin.

Podocalyxin is a CD34-related sialomucin that is expressed at high levels by podocytes, and also by mesothelial cells, vascular endothelia, platelets, and hematopoietic stem cells. To elucidate the function of podocalyxin, we generated podocalyxin-deficient (podxl(-/)-) mice by homologous recombination. Null mice exhibit profound defects in kidney development and die within 24 hours of birth with anuric renal failure. Although podocytes are present in the glomeruli of the podxl(-/)- mice, they fail to form foot processes and slit diaphragms and instead exhibit cell--cell junctional complexes (tight and adherens junctions). The corresponding reduction in permeable, glomerular filtration surface area presumably leads to the observed block in urine production. In addition, podxl(-/)- mice frequently display herniation of the gut (omphalocele), suggesting that podocalyxin may be required for retraction of the gut from the umbilical cord during development. Hematopoietic and vascular endothelial cells develop normally in the podocalyxin-deficient mice, possibly through functional compensation by other sialomucins (such as CD34). Our results provide the first example of an essential role for a sialomucin in development and suggest that defects in podocalyxin could play a role in podocyte dysfunction in renal failure and omphalocele in humans.

Characterization of prethymic progenitors within the chicken embryo.

The thymic primordium in both birds and mammals is first colonized by cells emerging from the intra-embryonic mesenchyme but the nature of these precursors is poorly understood. We demonstrate here an early embryonic day 7 prethymic population with T lymphoid potential. Our work is a phenotypic analysis of, to date, the earliest embryonic prethymic progenitors arising in the avian para-aortic area during ontogeny. The phenotype of these cells, expressing the cell surface molecules alpha2beta1 integrin, c-kit, thrombomucin/MEP21, HEMCAM and chL12, reflects functional properties required for cell adhesion, migration and growth factor responsiveness. Importantly, the presence of these antigens was found to correlate with the recolonization of the recipient thymus following intrathymic cell transfers. These intra-embryonic cells were also found to express the Ikaros transcription factor, the molecular function of which is considered to be prerequisite for embryonic lymphoid development.

Distinct C/EBP functions are required for eosinophil lineage commitment and maturation.

Hematopoietic differentiation involves the commitment of multipotent progenitors to a given lineage, followed by the maturation of the committed cells. To study the transcriptional events controlling these processes, we have investigated the role of C/EBP proteins in lineage choice of multipotent hematopoietic progenitors (MEPs) transformed by the E26 virus. We found that forced expression of either the alpha or beta isoforms of C/EBP in MEPs induced eosinophil differentiation and that in addition, C/EBPbeta could induce myeloid differentiation. Conversely, dominant-negative versions of C/EBPbeta inhibited myeloid differentiation. C/EBP-induced eosinophil differentiation could be separated into two distinct events, lineage commitment and maturation. Thus, eosinophils induced by transactivation-deficient C/EBPbeta alleles were found to be blocked in their maturation, whereas those expressing wild-type C/EBP proteins were not. Likewise, a 1-day activation of a conditional C/EBPbeta allele in multipotent progenitors led to the formation of immature eosinophils, whereas sustained activation produced mature eosinophils. These results show that C/EBP can induce both myeloid and eosinophil lineage commitment and that transactivation independent and dependent C/EBP functions are required during eosinophil lineage commitment and maturation, respectively.

Regulation of eosinophil-specific gene expression by a C/EBP-Ets complex and GATA-1.

The EOS47 antigen is an early and specific marker of eosinophil differentiation in the chicken haematopoietic system. To elucidate the transciptional events controlling commitment to the eosinophil lineage, we studied the regulation of the eosinophil-specific EOS47 promoter. This promoter is TATA-less, and binds trancription factors of the Ets, C/EBP, GATA and Myb families. These sites are contained within a 309 bp promoter fragment which is sufficient for specific high level transcription in an eosinophil cell line. Co-transfection experiments in Q2bn fibroblasts showed cooperative activation of the EOS47 proximal promoter by c-Myb, Ets-1/Fli-1, GATA-1 and C/EBPalpha. The Ets-1/Fli-1 and C/EBPalpha proteins were the most potent activators, and acted with high synergy through juxtaposed binding sites located approximately 60 bp upstream of the transcription start site. The Ets-1 and C/EBPalpha proteins were found to associate physically via their DNA-binding domains and to bind their combined binding site cooperatively. GATA-1 showed biphasic regulation of the EOS47 promoter, activating at low and repressing at high protein concentrations. These results demonstrate combinatorial activation of an eosinophil-specific promoter by ubiquitous and lineage-restricted haematopoietic transcription factors. They also indicate that direct interactions between C/EBPs and specific Ets family members, together with GATA-1, are important for eosinophil lineage determination.

Thrombomucin, a novel cell surface protein that defines thrombocytes and multipotent hematopoietic progenitors.

MEP21 is an avian antigen specifically expressed on the surface of Myb-Ets-transformed multipotent hematopoietic precursors (MEPs) and of normal thrombocytes. Using nanoelectrospray tandem mass spectrometry, we have sequenced and subsequently cloned the MEP21 cDNA and named the gene thrombomucin as it encodes a 571-amino acid protein with an extracellular domain typical of the mucin family of proteoglycans. Thrombomucin is distantly related to CD34, the best characterized and most used human hematopoietic stem cell marker. It is also highly homologous in its transmembrane/intracellular domain to podocalyxinlike protein-1, a rabbit cell surface glycoprotein of kidney podocytes. Single cell analysis of yolk sac cells from 3-d-old chick embryos revealed that thrombomucin is expressed on the surface of both lineage-restricted and multipotent progenitors. In the bone marrow, thrombomucin is also expressed on mono- and multipotent progenitors, showing an overlapping but distinct expression pattern from that of the receptor-type stem cell marker c-kit. These observations strengthen the notion that the Myb-Ets oncoprotein can induce the proliferation of thrombomucin-positive hematopoietic progenitors that have retained the capacity to differentiate along multiple lineages. They also suggest that thrombomucin and CD34 form a family of stem cell-specific proteins with possibly overlapping functions in early hematopoietic progenitors.

HEMCAM, an adhesion molecule expressed by c-kit+ hemopoietic progenitors.

We have characterized the adhesion molecule HEMCAM, which is expressed by hemopoietic progenitors of embryonic bone marrow. HEMCAM belongs to the immunoglobulin superfamily and consists of the V-V-C2-C2-C2 Ig domains. There are three mRNA splice variants. One has a short cytoplasmic tail; another has a long tail; while the third seems to lack transmembrane and cytoplasmic regions. Except for the NH2-terminal sequence, HEMCAM is identical to gicerin, a molecular involved in neurite outgrowth and Wilm's kidney tumor progression in the chicken and it is significantly homologous with MUC18 a molecule involved in melanoma progression and metastasis in human beings. In the bone marrow the HEMCAM+ cell population contains c-kit+ subsets. HEMCAM+ cells coexpressing the receptor tyrosine kinase c-kit give rise to T cells at a frequency of 0.17 when injected intrathymically in congenic animals. As HEMCAM+, c-kit+ cells differentiate into myeloid and erythroid CFU's the double-positive cell population seems to contain precursors for multiple lineages. HEMCAM promotes cell-cell adhesion of transfected cells. Cross-linking of murine HEMCAM leads to cell spreading of T-lymphocyte progenitors adhering to the vascular adhesion molecules, PECAM-1 and VCAM-1. Thus, HEMCAM is likely to be involved in cellular adhesion and homing processes.

Excision of Ets by an inducible site-specific recombinase causes differentiation of Myb-Ets-transformed hematopoietic progenitors.

BACKGROUND: The Myb-Ets protein encoded by the E26 acute avian leukemia virus is a paradigm for the function of fused transcriptional activator oncoproteins. Myb-Ets transforms hematopoietic progenitor cells (myb-Ets progenitors, MEPs) that can be induced to differentiate into eosinophilic and myeloid cells by the activation of pathways involving Ras and/or protein kinase C. The Ets portion of the fusion protein seems to be required to maintain the multipotency of MEPs: MEPs transformed with a temperature-sensitive E26 mutant with a lesion in Ets (ts 1.1) and shifted to the non-permissive temperature predominantly form erythroid cells, but also form eosinophilic and myeloid cells. This interpretation is complicated, however, by the observation that ts 1.1-transformed MEPs differ from MEPs transformed with wild-type E26 in that they expressed erythroid and eosinophil markers even at the permissive temperature. RESULTS: In order to alleviate the problems associated with the use of temperature-sensitive mutants we have designed a vector that allows the inducible deletion of the Ets domain. To this end, we introduced FLP recombinase target sites into the E26 virus on the 5' and 3' sides of Ets and included within the same retroviral vector sequences encoding and estrogen-dependent FLP recombinase. This construct, termed FRV-3, is capable of transforming cells to produce a phenotype indistinguishable from that of MEPs obtained with wild-type virus. Hormone treatment of MEPs transformed with FRV-3 induced erythroid differentiation in a subpopulation of the cells; this subpopulation was found to have completely excised ets. However, in contrast to previous results obtained with ts 1.1-transformed MEPs, no differentiation along the eosinophilic and myeloid lineages was seen in hormone-treated FRV-3-transformed MEPs. CONCLUSIONS: Our results demonstrate the feasibility of using a site-specific recombinase to excise a fused oncoprotein domain encoded by a retrovirus. More specifically, they show that the Ets portion of the Myb-Ets protein selectively inhibits differentiation of MEPs along the erythroid lineage, and suggests that Ets is also required for their differentiation along the eosinophil and, possibly, myeloid lineages.

The eosinophil-specific cell surface antigen, EOS47, is a chicken homologue of the oncofetal antigen melanotransferrin.

The EOS47 antigen is a 100-kD cell surface glycoprotein selectively expressed by avian retrovirus-transformed eosinophils and their precursors. We have purified the EOS47 protein to homogeneity and used peptide sequence information to clone EOS47-encoding cDNAs. The open reading frames from these cDNAs predict a 738 amino acid protein with homology to human melanotransferrin, a membrane-found, transferrin-like protein that is expressed at high levels by a subset of melanomas, tumor cell lines, fetal intestine, and liver, but not by most normal adult tissues. The predicted protein sequence of EOS47 displays a 61% sequence identity with melanotransferrin and conservation of all 28 cysteine residues, indicating a similar tertiary structure. The finding that EOS47 lacks several of the iron-coordinating amino acids present in all transferrins suggests that it may be impaired in its ability to bind iron. In nonhematopoietic tissues, EOS47 is expressed at high levels by epithelial brush borders of small intestine and kidney and at lower levels by cells lining the sinusoids of the liver. Within hematopoietic tissues, EOS47 is restricted to a subpopulation of cells (1% to 5%) in bone marrow and early spleen and fluorescence-activated cell sorting of EOS47+ cells leads to a dramatic ( > 30-fold) enrichment of peroxidase+ eosinophils. In contrast, peripheral blood eosinophils are EOS47-, suggesting that the antigen is expressed by newly formed eosinophils and that expression ceases shortly before these cells emigrate from the bone marrow into the peripheral blood. Our results show that melanotransferrin is a stage-specific marker of eosinophils and should be useful for their isolation and further characterization.

Integrin alpha 2 beta 1 mediates interactions between developing embryonic retinal cells and collagen.

In the developing nervous system, the extracellular matrix provides a source of extrinsic cues to guide determination of cell fate, neuroblast migration, axon outgrowth and synapse formation. In the neural retina, undifferentiated neuroepithelial precursor cells contact extracellular matrix that contains multiple collagen types. Collagens have been shown to support retinal cell adhesion and neurite outgrowth, but the integrin receptors mediating neuronal responses are not understood. Here we provide evidence that integrin alpha 2 beta 1 acts as a collagen receptor in the developing avian retina and examine its expression pattern. Using a recently described monoclonal antibody, MEP-17, alpha 2 protein was detected in the developing retina by immunofluorescence in tissue sections and dissociated cells, and by immunoprecipitation. At embryonic day 4 (E4), when the majority of retinal cells are undifferentiated neuroepithelial cells, alpha 2 immunoreactivity in sections was widespread and about half of cells dissociated in culture were alpha 2 positive. At E6, after the retinal ganglion cell layer had differentiated, immunoreactivity in sections decreased in the central, more developed portion of the retina and 25% of dissociated cells were alpha 2 positive. E6 retinal ganglion cells, identified by neurofilament immunoreactivity, did not express detectable alpha 2 immunoreactivity. Immunoprecipitation experiments using E6 extracts demonstrated that the alpha 2 subunit was paired with the beta 1 integrin subunit. By E12, alpha 2 immunoreactivity in sections was confined to the extreme peripheral retina, although the antigen may be masked since expression levels comparable to or slightly higher than E6 could be detected in dissociated cells and extracts. By employing function blocking antibodies, it was shown that alpha 2 beta 1 integrin is necessary for cell adhesion and process outgrowth by embryonic retinal cells on collagens I and IV. Although alpha 2 expression continued through E12, alpha 2 activity was down regulated with increasing embryonic age, since alpha 2-dependent adhesion and outgrowth declined. These data suggest a role for alpha 2 beta 1 in neuroepithelial cell interactions with collagen rather than for axon extension by retinal ganglion cells.

v-Myb DNA binding is required to block thrombocytic differentiation of Myb-Ets-transformed multipotent haematopoietic progenitors.

The E26 avian leukaemia virus encodes a fusion oncoprotein consisting of truncated versions of the c-Myb and c-Ets-1 transcription factors. When used to infect embryonic chicken haematopoietic cells two types of self-renewing progenitors are obtained, namely myeloblasts and 'MEPs' (Myb-Ets progenitors). In earlier work we have shown that myeloblasts transformed by the ts21 mutant of E26, which has a lesion in v-Myb, can be induced to differentiate into macrophages following shift to the non-permissive temperature. Here we show that the ts21 v-Myb is temperature sensitive for DNA binding in band shift experiments and that its inactivation in transformed MEPs induces their maturation into thrombocytes. The MEP transforming capacity of v-Myb is not confined to its fusion with v-Ets, as it is also seen with a virus that co-expresses tsMyb with v-ErbB. As with wild-type E26-transformed MEPs, ts21-transformed MEPs are multipotent, differentiating into eosinophils and myeloblasts following treatment with 12-O-tetradecanoylphorbol-13-acetate. In addition, ts21-transformed myeloblasts differentiate into macrophages when shifted to the non-permissive temperature. This shows that v-Myb blocks haematopoietic differentiation at two distinct stages. In contrast, v-Ets inactivation in MEPs transformed by a ts E26 mutant with a lesion in the corresponding oncoprotein leads to their differentiation into erythrocytes, myeloblasts and probably eosinophils. These data show that the two domains of Myb-Ets selectively affect decision making processes in different types and stages of haematopoietic cells.

A functional Ets DNA-binding domain is required to maintain multipotency of hematopoietic progenitors transformed by Myb-Ets.

Earlier work demonstrated that the Myb-Ets fusion protein of E26 avian leukemia virus induces the proliferation of multipotent hematopoietic progenitors (MEPs). These progenitors differentiate spontaneously at low frequencies along the erythroid lineage, and following the introduction of kinase/ras-type oncogenes or treatment with TPA, they are induced to differentiate along the myelomonocytic and eosinophilic lineages. Here, we show that the ts1.1 mutant of E26 encodes an Ets DNA-binding domain that is both defective and thermolabile for binding of specific DNA sequences. Correlating with this, ts1.1 MEP colonies transformed at the permissive temperature exhibit elevated levels of erythroid cells and eosinophils, whereas at the nonpermissive temperature they are induced to differentiate along the erythroid and myelomonocytic lineages and, to a lesser extent, along the eosinophil lineage. Induction of the former two lineages cannot be separated by pulse shift experiments and is essentially completed 2.5 days after temperature shift. Our results indicate that the Ets portion of the Myb-Ets fusion protein inhibits the lineage commitment of multipotent hematopoietic progenitors, probably via binding to regulatory DNA sequences of specific target genes.