Requirement for intron structures in activating the Cd8a locus.

During differentiation of CD4+CD8+ double-positive (DP) thymocytes into the CD4-CD8+ single-positive (CD8SP) thymocytes committed to the cytotoxic T cell lineage, Cd8a transcription is temporally terminated after positive selection and is subsequently reinitiated, a process known as coreceptor reversal. Despite the identification of a transcriptional enhancer in the Cd8a gene that directs reporter transgene expression specifically in CD8SP thymocytes, the molecular mechanisms controlling reactivation of the Cd8a gene are not fully understood. Here, we show that, after positive selection, hCD2 reporter expression from the Cd8a locus, which was generated by insertion of hCD2 cDNA into the first exon of the Cd8a gene, requires the incorporation of intron sequences into the hCD2 transcript. The presence of polyadenylation signals after hCD2 cDNA inhibited hCD2 expression in mature CD8+ T cells, whereas hCD2 expression in DP thymocytes recapitulated the Cd8a expression. Incorporation of the endogenous short intron structure and heterologous intron structure of the Cd4 locus restored hCD2 expression in mature CD8+ T cells in a variegated manner. Interestingly, stage-specific DNA demethylation was impaired in Cd8a reporter alleles that failed to express hCD2 in CD8+ T cells, and intron sequences lacking RNA splicing signals still restored hCD2 expression. These observations indicate that "intron-mediated enhancement" is involved in a stage-specific reactivation of the Cd8a locus harboring hCD2 cDNA. However, the Cd8a gene was transcribed in mature CD8+ T cells, albeit at a lower level, from a mutant Cd8a locus lacking intron structures, suggesting that protein-coding sequences in transcripts affect sensitivity to intron-mediated enhancement.

Unique N-terminal sequences in two Runx1 isoforms are dispensable for Runx1 function.

BACKGROUND: The Runt-related transcription factors (Runx) are a family of evolutionarily conserved transcriptional regulators that play multiple roles in the developmental control of various cell types. Among the three mammalian Runx proteins, Runx1 is essential for definitive hematopoiesis and its dysfunction leads to human leukemogenesis. There are two promoters, distal (P1) and proximal (P2), in the Runx1 gene, which produce two Runx1 isoforms with distinct N-terminal amino acid sequences, P1-Runx1 and P2-Runx1. However, it remains unclear whether P2-Runx specific N-terminal sequence have any specific function for Runx1 protein. RESULTS: To address the function of the P2-Runx1 isoform, we established novel mutant mouse models in which the translational initiation AUG (+1) codon for P2-Runx1 isoform was modulated. We found that a truncated P2-Runx1 isoform is translated from a downstream non-canonical AUG codon. Importantly, the truncated P2-Runx1 isoform is sufficient to support primary hematopoiesis, even in the absence of the P1-Runx1 isoform. Furthermore, the truncated P2-Runx1 isoform was able to restore defect in basophil development caused by loss of the P1-Runx1 isoform. The truncated P2-Runx1 isoform was more stable than the canonical P2-Runx1 isoform. CONCLUSIONS: Our results demonstrate that the N-terminal sequences specific for P2-Runx1 are dispensable for Runx1 function, and likely serve as a de-stabilization module to regulate Runx1 production.

Langerhans cell maturation is accompanied by induction of N-cadherin and the transcriptional regulators of epithelial-mesenchymal transition ZEB1/2.

Langerhans cells (LCs) are a unique subset of dendritic cells (DCs) that express epithelial adhesion molecules, allowing them to form contacts with epithelial cells and reside in epidermal/epithelial tissues. The dynamic regulation of epithelial adhesion plays a decisive role in the life cycle of LCs. It controls whether LCs remain immature and sessile within the epidermis or mature and egress to initiate immune responses. So far, the molecular machinery regulating epithelial adhesion molecules during LC maturation remains elusive. Here, we generated pure populations of immature human LCs in vitro to systematically probe for gene-expression changes during LC maturation. LCs down-regulate a set of epithelial genes including E-cadherin, while they upregulate the mesenchymal marker N-cadherin known to facilitate cell migration. In addition, N-cadherin is constitutively expressed by monocyte-derived DCs known to exhibit characteristics of both inflammatory-type and interstitial/dermal DCs. Moreover, the transcription factors ZEB1 and ZEB2 (ZEB is zinc-finger E-box-binding homeobox) are upregulated in migratory LCs. ZEB1 and ZEB2 have been shown to induce epithelial-to-mesenchymal transition (EMT) and invasive behavior in cancer cells undergoing metastasis. Our results provide the first hint that the molecular EMT machinery might facilitate LC mobilization. Moreover, our study suggests that N-cadherin plays a role during DC migration.

Molecular Analysis of Carbapenem and Aminoglycoside Resistance Genes in Carbapenem-Resistant Pseudomonas aeruginosa Clinical Strains: A Challenge for Tertiary Care Hospitals.

Carbapenem-resistant Pseudomonas aeruginosa (P. aeruginosa) strains have become a global threat due to their remarkable capability to survive and disseminate successfully by the acquisition of resistance genes. As a result, the treatment strategies have been severely compromised. Due to the insufficient available data regarding P. aeruginosa resistance from Pakistan, we aimed to investigate the resistance mechanisms of 249 P. aeruginosa strains by antimicrobial susceptibility testing, polymerase chain reaction for the detection of carbapenemases, aminoglycoside resistance genes, extended-spectrum beta-lactamases (ESBLs), sequence typing and plasmid typing. Furthermore, we tested silver nanoparticles (AgNPs) to evaluate their in vitro sensitivity against antimicrobial-resistant P. aeruginosa strains. We observed higher resistance against antimicrobials in the general surgery ward, general medicine ward and wound samples. Phenotypic carbapenemase-producer strains comprised 80.7% (201/249) with 89.0% (179/201) demonstrating genes encoding carbapenemases: blaNDM-1 (32.96%), blaOXA48 (37.43%), blaIMP (7.26%), blaVIM (5.03%), blaKPC-2 (1.12%), blaNDM-1/blaOXA48 (13.97%), blaOXA-48/blaVIM (1.68%) and blaVIM/blaIMP (0.56%). Aminoglycoside-modifying enzyme genes and 16S rRNA methylase variants were detected in 43.8% (109/249) strains: aac(6')-lb (12.8%), aac(3)-lla (12.0%), rmtB (21.1%), rmtC (11.0%), armA (12.8%), rmtD (4.6%), rmtF (6.4%), rmtB/aac(3)-lla (8.2%), rmtB/aac(6')-lla (7.3%) and rmtB/armA (3.6%). In total, 43.0% (77/179) of the strains coharbored carbapenemases and aminoglycoside resistance genes with 83.1% resistant to at least 1 agent in 3 or more classes and 16.9% resistant to every class of antimicrobials tested. Thirteen sequence types (STs) were identified: ST235, ST277, ST234, ST170, ST381, ST175, ST1455, ST1963, ST313, ST207, ST664, ST357 and ST348. Plasmid replicon types IncFI, IncFII, IncA/C, IncL/M, IncN, IncX, IncR and IncFIIK and MOB types F11, F12, H121, P131 and P3 were detected. Meropenem/AgNPs and Amikacin/AgNPs showed enhanced antibacterial activity. We reported the coexistence of carbapenemases and aminoglycoside resistance genes among carbapenem-resistant P. aeruginosa with diverse clonal lineages from Pakistan. Furthermore, we highlighted AgNP's potential role in handling future antimicrobial resistance concerns.

Exploring the associations between elevated plasma SP-D levels and OSCAR gene expression as potential biomarkers in patients with COPD: a cross-sectional study.

Background: Chronic obstructive pulmonary disease (COPD) imposes a substantial burden on patients and healthcare systems. Spirometry is the most widely used test to diagnose the disease; however, a surrogate marker is required to predict the disease pattern and progression. Objectives: The aim of the current study was to explore the association of elevated levels of plasma surfactant protein D (SP-D) with gene expression of osteoclast-associated receptor (OSCAR) and lung functions as potential diagnostic biomarkers of COPD. Methods: This cross-sectional study employed convenience sampling. As men compose the majority of patients in the outpatient department and with smoking being common among Pakistani men, choosing men offered a representative sample. Using a post-bronchodilator forced expiratory volume in the first second (FEV1) to a forced vital capacity (FVC) of less than 0.70 (FEV1/FVC <0.7), COPD patients were diagnosed on spirometry (n = 41). Controls were healthy individuals with FEV1/FVC >0.7 (n = 41). Plasma SP-D levels were measured using an enzyme-linked immunosorbent assay (ELISA). The gene expression of OSCAR was determined by real-time polymerase chain reaction (qPCR) and subsequently analyzed by the threshold cycle (Ct) method. Statistical Package for Social Sciences (SPSS) version 20 was used for statistical analysis. Results: The mean BMI of controls (25.66 ± 4.17 kg/m2) was higher than that of cases (23.49 ± 2.94 kg/m2 (p = .008)). The median age of controls was 49 years (interquartile range (IQR) 42.0-65.0 years) and that of cases was 65 years (IQR = 57.50-68.50). SP-D concentration was not significantly higher in COPD patients [4.96 ng/mL (IQR 3.26-7.96)] as compared to controls [3.64 ng/mL (IQR 2.60-8.59)] (p = .209). The forced expiratory ratio (FEV1/FVC) and FEV1 were related to gene expression of OSCAR (p = <.001). The gene expression of OSCAR was significantly related to SP-D (p = .034). A multiple regression model found FEV1 and FVC to have a significant effect on the gene expression of OSCAR (p-values <0.001 and 0.001, respectively). Conclusion: Gene expression of OSCAR was increased in COPD patients and related directly to SP-D levels and inversely to lung functions in cohort of this study, suggesting that OSCAR along with SP-D may serve as a diagnostic biomarker of COPD.

Association of Surfactant Protein D Single Nucleotide Polymorphisms rs721917, rs2243639, rs3088308 with Recurrent Aphthous Stomatitis in Pakistani Population.

Recurrent aphthous stomatitis (RAS) is a benign ulcerative condition, defined by the recurrent formation of non-contagious mucosal ulcers. Surfactant protein D (SP-D) is secreted frequently at surfaces exposed directly to body fluids. This study aims to investigate the association of SP-D single nucleotide polymorphisms (SNPs) with the onset of RAS. Blood samples from 212 subjects (106 cases/controls each) were collected during 2019 and genotyped for SP-D SNPs (rs721917, rs2243639, rs3088308) by polymerase chain reaction and restriction fragment length polymorphism followed by 12% polyacrylamide gel electrophoresis. Minor aphthous (75.5%) was the commonly observed ulcer type as compared to herpetiform (21.7%) and major aphthous ulcers (2.8%). A family history of RAS was reported in 70% of cases. RAS was found significantly associated with rs3088308 genotypes T/A (95% (Cl): 1.57-5.03, p = 0.0005), A/A (95% (Cl): 1.8-6.7, p = 0.0002), T-allele (95% (Cl): 1.09-2.36, p = 0.01), A-allele (95% (Cl): 1.42-3.91, p = 0.01), rs721917 genotype T/T (95% (Cl): 1.15-25.35, p = 0.03), and T-allele (95% (Cl): 1.28-3.10, p = 0.002). Female gender and obese body mass index (BMI) were significantly associated with rs3088308 genotypes T/A (95% (CI): 1.89-15.7, p = 0.001), T/T (95% (Cl): 1.52-11.9, p = 0.005), A-allele (95% (Cl): 1.65-7.58, p < 0.001), and T-allele (95% (Cl): 1.4-10.1, p <0.001) and rs721917 genotype T/T (95% (CI) = 1.3-33, p = 0.02), respectively. This study describes the association of SP-D SNPs (rs721917, rs3088308) with RAS in the Pakistani population.

Emergence of multidrug-resistant ST11 blaKPC-2 producing Klebsiella pneumoniae coharboring blaCTX-M and blaSHV in Pakistan.

INTRODUCTION: Carbapenemases are primarily responsible for the intensified spread of multidrug-resistant (MDR) K. pneumoniae by virtue of antibiotics overuse. Therefore, frequent investigation of high-risk clones especially from developing world is crucial to curtail global spread. METHODOLOGY: In this observational study, 107 K. pneumoniae were retrieved and confirmed genotypically from April 2018 to March 2020 from tertiary care hospitals in Lahore, Pakistan. Carbapenemases and extended-spectrum ß-lactamases were verified by Polymerase Chain Reaction and Sanger sequencing. Multilocus sequence typing and plasmid replicon typing were used to assign clonal lineages and plasmid replicons. RESULTS: Among the K. pneumoniae, 72.9% (78/107) strains were carbapenem resistant (CR) with 65.4% (51/78) exhibiting carbapenemase producing phenotype. Among CR K. pneumoniae 38.5% (30/78) strains exhibited the following carbapenemase genotypes: blaNDM-1 (26.7%, 8/30), blaOXA-48 (26.7%, 8/30), blaKPC-2 (20.0%, 6/30), blaVIM (10.0%, 3/30), blaNDM-1/blaOXA-48 (10.0%, 3/30), blaOXA-48/blaVIM (3.3%, 1/30) and blaOXA-48/blaIMP (3.3%, 1/30). Tigecycline and polymyxin-B retained susceptible profile. ß-lactam drugs showed intermediate to high resistance. The occurrence of CR K. pneumoniae infections was significantly associated with wound (39.7%, p = 0.0007), pus (38.5%, p = 0.009), general surgery (34.6%, p = 0.002) and intensive-care unit (26.9%, p = 0.04). blaKPC-2 producing K. pneumoniae coharboring blaCTX-M/blaSHV (66.7%) and blaCTX-M (33.3%) exhibited sequence type (ST) 258 (n = 4) and ST11 (n = 2) sequence types with IncFII, IncN, IncFIIA, IncL/M and IncFIIK plasmids. CONCLUSIONS: This is the first report describing the emergence of MDR blaKPC-2 producing K. pneumoniae ST11 coharboring blaCTX-M and blaSHV in Pakistan.

Estimation, Evaluation and Characterization of Carbapenem Resistance Burden from a Tertiary Care Hospital, Pakistan.

Carbapenem resistance has become major concern in healthcare settings globally; therefore, its monitoring is crucial for intervention efforts to halt resistance spread. During May 2019-April 2022, 2170 clinical strains were characterized for antimicrobial susceptibility, resistance genes, replicon and sequence types. Overall, 42.1% isolates were carbapenem-resistant, and significantly associated with Klebsiella pneumoniae (K. pneumoniae) (p = 0.008) and Proteus species (p = 0.043). Carbapenemases were detected in 82.2% of isolates, with blaNDM-1 (41.1%) associated with the ICU (p < 0.001), cardiology (p = 0.042), pediatric medicine (p = 0.013) and wound samples (p = 0.041); blaOXA-48 (32.6%) was associated with the ICU (p < 0.001), cardiology (p = 0.008), pediatric medicine (p < 0.001), general surgery (p = 0.001), general medicine (p = 0.005) and nephrology (p = 0.020); blaKPC-2 (5.5%) was associated with general surgery (p = 0.029); blaNDM-1/blaOXA-48 (11.4%) was associated with general surgery (p < 0.001), and wound (p = 0.002), urine (p = 0.003) and blood (p = 0.012) samples; blaOXA-48/blaVIM (3.1%) was associated with nephrology (p < 0.001) and urine samples (p < 0.001). Other detected carbapenemases were blaVIM (3.0%), blaIMP (2.7%), blaOXA-48/blaIMP (0.1%) and blaVIM/blaIMP (0.3%). Sequence type (ST)147 (39.7%) represented the most common sequence type identified among K. pneumoniae, along with ST11 (23.0%), ST14 (15.4%), ST258 (10.9%) and ST340 (9.6%) while ST405 comprised 34.5% of Escherichia coli (E. coli) isolates followed by ST131 (21.2%), ST101 (19.7%), ST10 (16.0%) and ST69 (7.4%). Plasmid replicon types IncFII, IncA/C, IncN, IncL/M, IncFIIA and IncFIIK were observed. This is first report describing the carbapenem-resistance burden and emergence of blaKPC-2-ST147, blaNDM-1-ST340 and blaNDM-1-ST14 in K. pneumoniae isolates and blaNDM-1-ST69 and blaNDM-1/blaOXA-48-ST69 in E. coli isolates coharboring extended-spectrum beta-lactamases (ESBLs) from Pakistan.

Characterization of Genomic Diversity among Carbapenem-Resistant Escherichia coli Clinical Isolates and Antibacterial Efficacy of Silver Nanoparticles from Pakistan.

The emergence of carbapenem-resistant Escherichia coli (E. coli) is considered an important threat to public health resulting in resistance accumulation due to antibiotics misuse and selection pressure. This warrants periodic efforts to investigate and develop strategies for infection control. A total of 184 carbapenem-resistant clinical strains of E. coli were characterized for resistance pattern, resistance genes, plasmids, sequence types and in vitro efficacy of silver nanoparticles (AgNPs). Carbapenem resistance was prevalent in E. coli isolated from female patients (64.7%), urine samples (40.8%) and surgical wards (32.1%). Polymyxin-B showed higher susceptibility. ESBLs and carbapenemases were produced in 179 and 119 isolates, respectively. Carbapenemase-encoding genes were observed among 104 strains with blaNDM-1 (45.1%), blaOXA-48 (27%), blaNDM-7 (3.8%), blaNDM-1/blaOXA-48 (15.4%), blaNDM-7/blaOXA-48 (2.9%), blaOXA-48/blaVIM (3.8%) and blaNDM-1/blaVIM (2%). ESBL resistance genes were detected in 147 isolates, namely blaSHV (24.9%), blaCTX-M (17.7%), blaTEM (4.8%), blaSHV/blaCTX-M (29.2%), blaSHV/blaTEM (15%) and blaCTX-M/blaTEM (8.8%). ST405 (44.4%) and ST131 (29.2%) were more frequent sequence types with ST101 (9.7%), ST10 (9.7%) and ST648 (7%). The replicon types IncFII, IncFIIK, IncA/C, IncN and IncL/M were detected. The combination of MEM/AgNPs remained effective against carbapenemase-positive E. coli. We reported genetically diverse E. coli strains coharboring carbapenemases/ESBLs from Pakistan. Moreover, this study highlights the enhanced antibacterial activity of MEM/AgNPs and may be used to manage bacterial infections.

Novel Carbapenem-Resistant Klebsiella pneumoniae ST147 Coharboring bla NDM-1, bla OXA-48 and Extended-Spectrum ß-Lactamases from Pakistan.

PURPOSE: The emergence of multidrug-resistant Klebsiella pneumoniae (K. pneumoniae) is associated with the acquisition of multiple carbapenemases. Their clonal spread is a worldwide concern due to their critical role in nosocomial infections. Therefore, the identification of high-risk clones with antibiotic resistance genes is very crucial for controlling its global spread. MATERIALS AND METHODS: A total of 227 K. pneumoniae strains collected during April 2018 to November 2019 were confirmed by PCR. Carbapenemases and extended-spectrum ß-lactamases (ESBL) were detected phenotypically. Confirmation of carbapenemases was carried out by PCR and Sanger sequencing. The clonal lineages were assigned to selected isolates by multilocus sequence typing (MLST), and the plasmid analysis was done by PCR-based detection of the plasmid replicon typing. RESULTS: Of the total K. pneumoniae, 117 (51.5%) were carbapenem resistant (CRKP) and 140 (61.7%) were identified as ESBL producers. Intermediate to high resistance was detected in the tested ß-lactam drugs while polymyxin-B and tigecycline were found to be susceptible. Among CRKP, 91 (77.8%) isolates were detected as carbapenemase producing, while 55 (47%) were positive for bla NDM-1 23.9% (n=28), bla OXA-48 22.2% (n=26) and bla VIM 0.85% (n=1) while 12.7% (n=7) carried both bla NDM-1 and bla OXA-48 genes. The CRKP coharboring bla NDM-1 and bla OXA-48 genes (n=7) were positive for bla CTX-M bla SHV (n=3), bla SHV (n=1) and bla CTX-M (n=3). The novel CRKP with the coexistence of bla NDM-1, bla OXA-48, bla CTX-M and bla SHV genes were associated with the high-risk clone ST147 (n=5) and ST11 (n=2). The assigned replicon types were IncL/M, IncFII, IncA/C and IncH1. CONCLUSION: This is the first report of the coexistence of bla NDM-1, bla OXA-48, bla CTX-M and bla SHV genes on a high-risk lineage ST147 from Pakistan. This study highlights the successful dissemination of carbapenemase resistance genes in the high-risk clones that emphasizes the importance of monitoring and controlling the spread of these diverse clones globally.

Runx-dependent and silencer-independent repression of a maturation enhancer in the Cd4 gene.

An intronic silencer, S4, in the Cd4 gene has been shown to be responsible for the helper-lineage-specific expression of CD4; S4 requires Runx complex binding to exert its silencer function against the enhancer-mediated Cd4 activation by modulating the epigenetic state of the Cd4 gene. Here we identify a late-acting maturation enhancer. Bcl11b plays essential roles for activation of both the early-acting proximal enhancer and maturation enhancer of Cd4. Notably, Runx complexes suppress these enhancers by distinct mechanisms. Whereas repression of the proximal enhancer depends on the S4 silencer, the maturation enhancer is repressed by Runx in the absence of S4. Moreover, ThPOK, known to antagonize S4-mediated Cd4 repression, assists Runx complexes to restrain maturation enhancer activation. Distinct modes of S4 silencer action upon distinct enhancers thus unravel a pathway that restricts CD4 expression to helper-lineage cells by silencer-independent and Runx-dependent repression of maturation enhancer activity in cytotoxic-lineage cells.

Essential Roles of SATB1 in Specifying T Lymphocyte Subsets.

T cell receptor (TCR) signaling by MHC class I and II induces thymocytes to acquire cytotoxic and helper fates via the induction of Runx3 and ThPOK transcription factors, respectively. The mechanisms by which TCR signaling is translated into transcriptional programs for each cell fate remain elusive. Here, we show that, in post-selection thymocytes, a genome organizer, SATB1, activates genes for lineage-specifying factors, including ThPOK, Runx3, CD4, CD8, and Treg factor Foxp3, via regulating enhancers in these genes in a locus-specific manner. Indeed, SATB1-deficient thymocytes are partially re-directed into inappropriate T lineages after both MHC class I- and II-mediated selection, and they fail to generate NKT and Treg subsets. Despite its essential role in activating enhancers for the gene encoding ThPOK in TCR-signaled thymocytes, SATB1 becomes dispensable for maintaining ThPOK in CD4+ T cells. Collectively, our findings demonstrate that SATB1 shapes the primary T cell pool by directing lineage-specific transcriptional programs in the thymus.

A novel Cd8-cis-regulatory element preferentially directs expression in CD44hiCD62L+ CD8+ T cells and in CD8αα+ dendritic cells.

CD8 coreceptor expression is dynamically regulated during thymocyte development and is tightly controlled by the activity of at least 5 different cis-regulatory elements. Despite the detailed characterization of the Cd8 loci, the regulation of the complex expression pattern of CD8 cannot be fully explained by the activity of the known Cd8 enhancers. In this study, we revisited the Cd8ab gene complex with bioinformatics and transgenic reporter gene expression approaches to search for additional Cd8 cis-regulatory elements. This led to the identification of an ECR (ECR-4), which in transgenic reporter gene expression assays, directed expression preferentially in CD44(hi)CD62L(+) CD8(+) T cells, including innate-like CD8(+) T cells. ECR-4, designated as Cd8 enhancer E8VI, was bound by Runx/CBFß complexes and Bcl11b, indicating that E8VI is part of the cis-regulatory network that recruits transcription factors to the Cd8ab gene complex in CD8(+) T cells. Transgenic reporter expression was maintained in LCMV-specific CD8(+) T cells upon infection, although short-term, in vitro activation led to a down-regulation of E8VI activity. Finally, E8VI directed transgene expression also in CD8αα(+) DCs but not in CD8αα-expressing IELs. Taken together, we have identified a novel Cd8 enhancer that directs expression in CD44(hi)CD62L(+) CD8(+) T cells, including innate-like and antigen-specific effector/memory CD8(+) T cells and in CD8αα(+) DCs, and thus, our data provide further insight into the cis-regulatory networks that control CD8 expression.

ß-Catenin promotes the differentiation of epidermal Langerhans dendritic cells.

The epithelial signaling protein and transcriptional regulator ß-catenin has recently been implicated in hematopoietic dendritic cell (DC) differentiation as well as in DC-mediated tolerance. We here observed that epidermal Langerhans cells (LCs) but not interstitial/dermal DCs express detectable ß-catenin. LCs are unique among the DC family members in that LC networks critically depend on epithelial adhesion molecules as well as on the cytokine transforming growth factor-ß1 (TGF-ß1). However, despite the important functions of LCs in the immune system, the molecular mechanisms governing LC differentiation and maintenance remain poorly defined. We found that TGF-ß1 induces ß-catenin in progenitor cells undergoing LC differentiation and that ß-catenin promotes LC differentiation. Vitamin D, another epidermal signal, enhanced TGF-ß1-mediated ß-catenin induction and promoted the expression of multiple epithelial genes by LCs. Moreover, full-length vitamin D receptor (VDR) promoted, whereas a truncated VDR diminished, the positive effects of ectopic ß-catenin on LC differentiation. Therefore, we here identified ß-catenin as a positive regulator of LC differentiation in response to TGF-ß1 and identified a functional interaction between ß-catenin and VDR in these cells.

Identification of bone morphogenetic protein 7 (BMP7) as an instructive factor for human epidermal Langerhans cell differentiation.

Human Langerhans cell (LC) precursors populate the epidermis early during prenatal development and thereafter undergo massive proliferation. The prototypic antiproliferative cytokine TGF-ß1 is required for LC differentiation from human CD34(+) hematopoietic progenitor cells and blood monocytes in vitro. Similarly, TGF-ß1 deficiency results in LC loss in vivo. However, immunohistology studies revealed that human LC niches in early prenatal epidermis and adult basal (germinal) keratinocyte layers lack detectable TGF-ß1. Here we demonstrated that these LC niches express high levels of bone morphogenetic protein 7 (BMP7) and that Bmp7-deficient mice exhibit substantially diminished LC numbers, with the remaining cells appearing less dendritic. BMP7 induces LC differentiation and proliferation by activating the BMP type-I receptor ALK3 in the absence of canonical TGF-ß1-ALK5 signaling. Conversely, TGF-ß1-induced in vitro LC differentiation is mediated via ALK3; however, co-induction of ALK5 diminished TGF-ß1-driven LC generation. Therefore, selective ALK3 signaling by BMP7 promotes high LC yields. Within epidermis, BMP7 shows an inverse expression pattern relative to TGF-ß1, the latter induced in suprabasal layers and up-regulated in outer layers. We observed that TGF-ß1 inhibits microbial activation of BMP7-generated LCs. Therefore, TGF-ß1 in suprabasal/outer epidermal layers might inhibit LC activation, resulting in LC network maintenance.

Identification of Axl as a downstream effector of TGF-ß1 during Langerhans cell differentiation and epidermal homeostasis.

Transforming growth factor-ß1 (TGF-ß1) is a fundamental regulator of immune cell development and function. In this study, we investigated the effects of TGF-ß1 on the differentiation of human Langerhans cells (LCs) and identified Axl as a key TGF-ß1 effector. Axl belongs to the TAM (Tyro3, Axl, and Mer) receptor tyrosine kinase family, whose members function as inhibitors of innate inflammatory responses in dendritic cells and are essential to the prevention of lupus-like autoimmunity. We found that Axl expression is induced by TGF-ß1 during LC differentiation and that LC precursors acquire Axl early during differentiation. We also describe prominent steady-state expression as well as inflammation-induced activation of Axl in human epidermal keratinocytes and LCs. TGF-ß1-induced Axl enhances apoptotic cell (AC) uptake and blocks proinflammatory cytokine production. The antiinflammatory role of Axl in the skin is reflected in a marked impairment of the LC network preceding spontaneous skin inflammation in mutant mice that lack all three TAM receptors. Our findings highlight the importance of constitutive Axl expression to tolerogenic barrier immunity in the epidermis and define a mechanism by which TGF-ß1 enables silent homeostatic clearing of ACs to maintain long-term self-tolerance.

Identification of TROP2 (TACSTD2), an EpCAM-like molecule, as a specific marker for TGF-ß1-dependent human epidermal Langerhans cells.

Langerin (CD207) expression is a hallmark of epidermal Langerhans cells (LCs); however, CD207(+) cells comprise several functional subsets. Murine studies showed that epidermal, but not dermal, CD207(+) cells require transforming growth factor-ß 1 (TGF-ß1) for development, whereas human data are lacking. Using gene profiling, we found that the surface molecule TROP2 (TACSTD2) is strongly and rapidly induced during TGF-ß1-dependent LC commitment of human CD34(+) hematopoietic progenitor cells or monocytes. TROP2 is conserved between mouse and human, and shares substantial amino-acid identity with EpCAM, a marker for murine epidermal LCs. To our knowledge, neither TROP2 nor EpCAM expression has been analyzed in human dendritic cell (DC) subsets. We found that (i) all human epidermal LCs are TROP2(+)EpCAM(+); (ii) human dermis lacks CD207(+)EpCAM(-) or CD207(+)TROP2(-) DCs, i.e., equivalents of murine dermal CD207(+) DCs; and (iii) pulmonary CD207(+) cells are TROP2(-)EpCAM(-). Moreover, although EpCAM was broadly expressed by pulmonary and intestinal epithelial cells, as well as by bone marrow erythroid progenitor cells, these cells lacked TROP2. However, although TROP2 is expressed by human LCs as well as by human and murine keratinocytes, most murine LCs, except of a small subset, lacked TROP2. Therefore, TROP2 is a marker for human TGF-ß1-dependent epidermal LCs.