The impact of genomic distance on enhancer-promoter interactions at the CFTR locus.

We identified and characterized multiple cell-type selective enhancers of the CFTR gene promoter in previous work and demonstrated active looping of these elements to the promoter. Here we address the impact of genomic spacing on these enhancer:promoter interactions and on CFTR gene expression. Using CRISPR/Cas9, we generated clonal cell lines with deletions between the -35 kb airway enhancer and the CFTR promoter in the 16HBE14o- airway cell line, or between the intron 1 (185 + 10 kb) intestinal enhancer and the promoter in the Caco2 intestinal cell line. The effect of these deletions on CFTR transcript abundance, as well as the 3D looping structure of the locus was investigated in triplicate clones of each modification. Our results indicate that both small and larger deletions upstream of the promoter can perturb CFTR expression and -35 kb enhancer:promoter interactions in the airway cells, though the larger deletions are more impactful. In contrast, the small intronic deletions have no effect on CFTR expression and intron 1 enhancer:promoter interactions in the intestinal cells, whereas larger deletions do. Clonal variation following a specific CFTR modification is a confounding factor particularly in 16HBE14o- cells.

Stellate cells are in utero markers of pancreatic disease in cystic fibrosis.

BACKGROUND: Pancreatic fibrosis is an early diagnostic feature of the common inherited disorder cystic fibrosis (CF). Many people with CF (pwCF) are pancreatic insufficient from birth and the replacement of acinar tissue with cystic lesions and fibrosis is a progressive phenotype that may later lead to diabetes. Little is known about the initiating events in the fibrotic process though it may be a sequela of inflammation in the pancreatic ducts resulting from loss of CFTR impairing normal fluid secretion. Here we use a sheep model of CF (CFTR-/-) to examine the evolution of pancreatic disease through gestation. METHODS: Fetal pancreas was collected at six time points from 50-days of gestation through to term, which is equivalent to ~ 13 weeks to term in human. RNA was extracted from tissue for bulk RNA-seq and single cells were prepared from 80-day, 120-day and term samples for scRNA-seq. Data were validated by immunochemistry. RESULTS: Transcriptomic evidence from bulk RNA-seq showed alterations in the CFTR-/- pancreas by 65-days of gestation, which are accompanied by marked pathological changes by 80-days of gestation. These include a fibrotic response, confirmed by immunostaining for COL1A1, αSMA and SPARC, together with acinar loss. Moreover, using scRNA-seq we identify a unique cell population that is significantly overrepresented in the CFTR-/- animals at 80- and 120-days gestation, as are stellate cells at term. CONCLUSION: The transcriptomic changes and cellular imbalance that we observe likely have pivotal roles in the evolution of CF pancreatic disease and may provide therapeutic opportunities to delay or prevent pancreatic destruction in CF.

An ectopic enhancer restores CFTR expression through de novo chromatin looping.

Transcription of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is regulated by both ubiquitous and cell-type selective cis-regulatory elements (CREs). These CREs include extragenic and intronic enhancers that bind lineage-specific transcription factors, and architectural protein-marked structural elements. Deletion of the airway-selective -35 kb enhancer in 16HBE14o- lung epithelial cells was shown earlier to disrupt normal enhancer-promoter looping at the CFTR locus and nearly abolish its expression. Using a 16HBE14o- clone that lacks the endogenous -35 kb CRE, we explore the impact of relocating the functional core of this element to an ectopic site in intron 1. The -35 kb sequence establishes a de novo enhancer signature in chromatin at the insertion site, and augments CFTR expression, albeit not fully restoring WT levels. The relocated -35 kb enhancer also initiates de novo chromatin contacts with the CFTR promoter and other known CFTR CREs. These results are broadly relevant to therapeutic gene editing.

Transcriptomic analysis of lung development in wildtype and CFTR-/- sheep suggests an early inflammatory signature in the CF distal lung.

The precise molecular events initiating human lung disease are often poorly characterized. Investigating prenatal events that may underlie lung disease in later life is challenging in man, but insights from the well-characterized sheep model of lung development are valuable. Here, we determine the transcriptomic signature of lung development in wild-type sheep (WT) and use a sheep model of cystic fibrosis (CF) to characterize disease associated changes in gene expression through the pseudoglandular, canalicular, saccular, and alveolar stages of lung growth and differentiation. Using gene ontology process enrichment analysis of differentially expressed genes at each developmental time point, we define changes in biological processes (BP) in proximal and distal lung from WT or CF animals. We also compare divergent BP in WT and CF animals at each time point. Next, we establish the developmental profile of key genes encoding components of ion transport and innate immunity that are pivotal in CF lung disease and validate transcriptomic data by RT-qPCR. Consistent with the known pro-inflammatory phenotype of the CF lung after birth, we observe upregulation of inflammatory response processes in the CF sheep distal lung during the saccular stage of prenatal development. These data suggest early commencement of therapeutic regimens may be beneficial.

Dual SMAD inhibition enhances the longevity of human epididymis epithelial cells.

Primary human epididymis epithelial (HEE) cells are valuable reagents for functional studies on the human epididymis. We used them previously to determine the transcriptional networks that establish cell identity along the length of the epididymis from caput, corpus, and cauda. These studies on HEE cells and organoids derived from them revealed important cellular properties. However, similar to other primary cells, HEE cells undergo replicative senescence and de-differentiation in culture. A cocktail of small molecules was shown elsewhere to extend longevity of epithelial basal cells. The components included transforming growth factor ß (TGF-ß)/bone morphogenetic protein (BMP) antagonists, WNT agonist, and Rho-associated and coiled-coil containing protein kinase (ROCK) inhibitor (ROCKi), which together prevented the senescence-related upregulation of TGF-ß signaling pathway members. Here, we treat HEE cells with the same cocktail and observed enhanced replicative potential and prolonged expression of markers of HEE differentiation. This treatment expands the differentiated HEE cell population available from individual epididymis tissue samples that can be used for molecular, cellular, and functional studies.

Cross-talk between enhancers, structural elements and activating transcription factors maintains the 3D architecture and expression of the CFTR gene.

Robust protocols to examine 3D chromatin structure have greatly advanced knowledge of gene regulatory mechanisms. Here we focus on the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which provides a paradigm for validating models of gene regulation built upon genome-wide analysis. We examine the mechanisms by which multiple cis-regulatory elements (CREs) at the CFTR gene coordinate its expression in intestinal epithelial cells. Using CRISPR/Cas9 to remove CREs, individually and in tandem, followed by assays of gene expression and higher-order chromatin structure (4C-seq), we reveal the cross-talk and dependency of two cell-specific intronic enhancers. The results suggest a mechanism whereby the locus responds when CREs are lost, which may involve activating transcription factors such as FOXA2. Also, by removing the 5' topologically-associating domain (TAD) boundary, we illustrate its impact on CFTR gene expression and architecture. These data suggest a multi-layered regulatory hierarchy that is highly sensitive to perturbations.

Krüppel-like factor 5 regulates wound repair and the innate immune response in human airway epithelial cells.

A complex network of transcription factors regulates genes involved in establishing and maintaining key biological properties of the human airway epithelium. However, detailed knowledge of the contributing factors is incomplete. Here we characterize the role of Krüppel-like factor 5 (KLF5), in controlling essential pathways of epithelial cell identity and function in the human lung. RNA-seq following siRNA-mediated depletion of KLF5 in the Calu-3 lung epithelial cell line identified significant enrichment of genes encoding chemokines and cytokines involved in the proinflammatory response and also components of the junctional complexes mediating cell adhesion. To determine direct gene targets of KLF5, we defined the cistrome of KLF5 using ChIP-seq in both Calu-3 and 16HBE14o- lung epithelial cell lines. Occupancy site concordance analysis revealed that KLF5 colocalized with the active histone modification H3K27ac and also with binding sites for the transcription factor CCAAT enhancer-binding protein beta (C/EBPß). Depletion of KLF5 increased both the expression and secretion of cytokines including IL-1ß, a response that was enhanced following exposure to Pseudomonas aeruginosa lipopolysaccharide. Calu-3 cells exhibited faster rates of repair after KLF5 depletion compared with negative controls in wound scratch assays. Similarly, CRISPR-mediated KLF5-null 16HBE14o- cells also showed enhanced wound closure. These data reveal a pivotal role for KLF5 in coordinating epithelial functions relevant to human lung disease.

BACH1, the master regulator of oxidative stress, has a dual effect on CFTR expression.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene lies within a topologically associated domain (TAD) in which multiple cis-regulatory elements (CREs) and transcription factors (TFs) regulate its cell-specific expression. The CREs are recruited to the gene promoter by a looping mechanism that depends upon both architectural proteins and specific TFs. An siRNA screen to identify TFs coordinating CFTR expression in airway epithelial cells suggested an activating role for BTB domain and CNC homolog 1 (BACH1). BACH1 is a ubiquitous master regulator of the cellular response to oxidative stress. Here, we show that BACH1 may have a dual effect on CFTR expression by direct occupancy of CREs at physiological oxygen (∼8%), while indirectly modulating expression under conditions of oxidative stress. Hence BACH1, can activate or repress the same gene, to fine tune expression in response to environmental cues such as cell stress. Furthermore, our 4C-seq data suggest that BACH1 can also directly regulate CFTR gene expression by modulating locus architecture through occupancy at known enhancers and structural elements, and depletion of BACH1 alters the higher order chromatin structure.

Looping of upstream cis-regulatory elements is required for CFTR expression in human airway epithelial cells.

The CFTR gene lies within an invariant topologically associated domain (TAD) demarcated by CTCF and cohesin, but shows cell-type specific control mechanisms utilizing different cis-regulatory elements (CRE) within the TAD. Within the respiratory epithelium, more than one cell type expresses CFTR and the molecular mechanisms controlling its transcription are likely divergent between them. Here, we determine how two extragenic CREs that are prominent in epithelial cells in the lung, regulate expression of the gene. We showed earlier that these CREs, located at -44 and -35 kb upstream of the promoter, have strong cell-type-selective enhancer function. They are also responsive to inflammatory mediators and to oxidative stress, consistent with a key role in CF lung disease. Here, we use CRISPR/Cas9 technology to remove these CREs from the endogenous locus in human bronchial epithelial cells. Loss of either site extinguished CFTR expression and abolished long-range interactions between these sites and the gene promoter, suggesting non-redundant enhancers. The deletions also greatly reduced promoter interactions with the 5' TAD boundary. We show substantial recruitment of RNAPII to the -35 kb element and identify CEBPß as a key activator of airway expression of CFTR, likely through occupancy at this CRE and the gene promoter.

OTX2 regulates CFTR expression during endoderm differentiation and occupies 3' cis-regulatory elements.

BACKGROUND: Cell-specific and developmental mechanisms contribute to expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene; however, its developmental regulation is poorly understood. Here we use human induced pluripotent stem cells differentiated into pseudostratified airway epithelial cells to study these mechanisms. RESULTS: Changes in gene expression and open chromatin profiles were investigated by RNA-seq and ATAC-seq, and revealed that alterations in CFTR expression are associated with differences in stage-specific open chromatin. Additionally, two novel open chromatin regions, at +19.6 kb and +22.6 kb 3' to the CFTR translational stop signal, were observed in definitive endoderm (DE) cells, prior to an increase in CFTR expression in anterior foregut endoderm (AFE) cells. Chromatin studies in DE and AFE cells revealed enrichment of active enhancer marks and occupancy of OTX2 at these sites in DE cells. Loss of OTX2 in DE cells alters histone modifications across the CFTR locus and results in a 2.5-fold to 5-fold increase in CFTR expression. However, deletion of the +22.6 kb site alone does not affect CFTR expression in DE or AFE cells. CONCLUSIONS: These results suggest that a network of interacting cis-regulatory elements recruit OTX2 to the locus to impact CFTR expression during early endoderm differentiation.

A functional genomics approach to investigate the differentiation of iPSCs into lung epithelium at air-liquid interface.

The availability of robust protocols to differentiate induced pluripotent stem cells (iPSCs) into many human cell lineages has transformed research into the origins of human disease. The efficacy of differentiating iPSCs into specific cellular models is influenced by many factors including both intrinsic and extrinsic features. Among the most challenging models is the generation of human bronchial epithelium at air-liquid interface (HBE-ALI), which is the gold standard for many studies of respiratory diseases including cystic fibrosis. Here, we perform open chromatin mapping by ATAC-seq and transcriptomics by RNA-seq in parallel, to define the functional genomics of key stages of the iPSC to HBE-ALI differentiation. Within open chromatin peaks, the overrepresented motifs include the architectural protein CTCF at all stages, while motifs for the FOXA pioneer and GATA factor families are seen more often at early stages, and those regulating key airway epithelial functions, such as EHF, are limited to later stages. The RNA-seq data illustrate dynamic pathways during the iPSC to HBE-ALI differentiation, and also the marked functional divergence of different iPSC lines at the ALI stages of differentiation. Moreover, a comparison of iPSC-derived and lung donor-derived HBE-ALI cultures reveals substantial differences between these models.

The FOXA1 transcriptional network coordinates key functions of primary human airway epithelial cells.

The differentiated functions of the human airway epithelium are coordinated by a complex network of transcription factors. These include the pioneer factors Forkhead box A1 and A2 (FOXA1 and FOXA2), which are well studied in several tissues, but their role in airway epithelial cells is poorly characterized. Here, we define the cistrome of FOXA1 and FOXA2 in primary human bronchial epithelial (HBE) cells by chromatin immunoprecipitation with deep-sequencing (ChIP-seq). Next, siRNA-mediated depletion of each factor is used to investigate their transcriptome by RNA-seq. We found that, as predicted from their DNA-binding motifs, genome-wide occupancy of the two factors showed substantial overlap; however, their global impact on gene expression differed. FOXA1 is an abundant transcript in HBE cells, while FOXA2 is expressed at low levels, and both these factors likely exhibit autoregulation and cross-regulation. FOXA1 regulated loci are involved in cell adhesion and the maintenance of epithelial cell identity, particularly through repression of genes associated with epithelial to mesenchymal transition (EMT). FOXA1 also directly targets other transcription factors with a known role in the airway epithelium such as SAM-pointed domain-containing Ets-like factor (SPDEF). The intersection of the cistrome and transcriptome for FOXA1 revealed enrichment of genes involved in epithelial development and tissue morphogenesis. Moreover, depletion of FOXA1 was shown to reduce the transepithelial resistance of HBE cells, confirming the role of this factor in maintaining epithelial barrier integrity.

Investigating adverse genomic and regulatory changes caused by replacement of the full-length CFTR cDNA using Cas9 and AAV.

A "universal strategy" replacing the full-length CFTR cDNA may treat >99% of people with cystic fibrosis (pwCF), regardless of their specific mutations. Cas9-based gene editing was used to insert the CFTR cDNA and a truncated CD19 (tCD19) enrichment tag at the CFTR locus in airway basal stem cells. This strategy restores CFTR function to non-CF levels. Here, we investigate the safety of this approach by assessing genomic and regulatory changes after CFTR cDNA insertion. Safety was first assessed by quantifying genetic rearrangements using CAST-seq. After validating restored CFTR function in edited and enriched airway cells, the CFTR locus open chromatin profile was characterized using ATAC-seq. The regenerative potential and differential gene expression in edited cells was assessed using scRNA-seq. CAST-seq revealed a translocation in ∼0.01% of alleles primarily occurring at a nononcogenic off-target site and large indels in 1% of alleles. The open chromatin profile of differentiated airway epithelial cells showed no appreciable changes, except in the region corresponding to the CFTR cDNA and tCD19 cassette, indicating no detectable changes in gene regulation. Edited stem cells produced the same types of airway cells as controls with minimal alternations in gene expression. Overall, the universal strategy showed minor undesirable genomic changes.

A nociceptive amygdala-striatal pathway for chronic pain aversion.

The basolateral amygdala (BLA) is essential for assigning positive or negative valence to sensory stimuli. Noxious stimuli that cause pain are encoded by an ensemble of nociceptive BLA projection neurons (BLAnoci ensemble). However, the role of the BLAnoci ensemble in mediating behavior changes and the molecular signatures and downstream targets distinguishing this ensemble remain poorly understood. Here, we show that the same BLAnoci ensemble neurons are required for both acute and chronic neuropathic pain behavior. Using single nucleus RNA-sequencing, we characterized the effect of acute and chronic pain on the BLA and identified enrichment for genes with known functions in axonal and synaptic organization and pain perception. We thus examined the brain-wide targets of the BLAnoci ensemble and uncovered a previously undescribed nociceptive hotspot of the nucleus accumbens shell (NAcSh) that mirrors the stability and specificity of the BLAnoci ensemble and is recruited in chronic pain. Notably, BLAnoci ensemble axons transmit acute and neuropathic nociceptive information to the NAcSh, highlighting this nociceptive amygdala-striatal circuit as a unique pathway for affective-motivational responses across pain states.

Functional genomics of the human epididymis: Further characterization of efferent ducts and model systems by single-cell RNA sequencing analysis.

BACKGROUND: The human epididymis is poorly studied due to the lack of availability of tissue samples. Our understanding of its structure and function depends upon anatomical and histological observations of archived material. OBJECTIVES: Here we used single-cell RNA sequencing (scRNA-seq) technologies to elucidate the identity of cells within the human efferent ducts (EDs) and compared them to caput epididymis cells. We also compared the cellularity of primary tissues with those of 2D and 3D (organoid) culture models used for functional studies. MATERIALS AND METHODS: Human epididymis tissue was dissected to separate different anatomical regions and digested to release single cells for processing on the 10X Genomics Chromium platform. Primary human epididymis epithelial (HEE) cells and HEE organoids were grown as described previously and subjected to scRNA-seq. scRNA-seq data were processed by standard bioinformatics pipelines and used for comparative analysis. RESULTS: We define the cell types in the EDs which include specialized epithelial cells, connective tissue stromal cells, vascular endothelial cells, smooth muscle cells, and immune cells, but lack basal cells that are seen in the caput epididymis. Furthermore, we identify a sub-population of epithelial cells which have marker genes found in the bladder and urothelium. Comparative genomics analysis of the 2D and 3D culture models shows cellular identities adapted to the culture environment while still maintaining similarity to the primary tissue. DISCUSSION: Our data suggest that EDs are lined with a transitional epithelium, which like the urothelium is able to stretch and contract depending on luminal volume. This is consistent with its primary role in seminal fluid resorption and sperm concentration. Moreover, we describe the cellularity of models to study the human epididymis epithelium in vitro. CONCLUSION: Single-cell RNA-seq data from the human epididymis make a valuable contribution to our understanding of this highly specialized organ.

Cellular heterogeneity in the 16HBE14o- airway epithelial line impacts biological readouts.

The airway epithelial cell line, 16HBE14o- , is an important cell model for studying airway disease. 16HBE14o- cells were originally generated from primary human bronchial epithelial cells by SV40-mediated immortalization, a process that is associated with genomic instability through long-term culture. Here, we explore the heterogeneity of these cells, with respect to expression of the cystic fibrosis transmembrane conductance regulator (CFTR) transcript and protein. We isolate clones of 16HBE14o- with stably higher and lower levels of CFTR in comparison to bulk 16HBE14o- , designated CFTRhigh and CFTRlow . Detailed characterization of the CFTR locus in these clones by ATAC-seq and 4C-seq showed open chromatin profiles and higher order chromatin structure that correlate with CFTR expression levels. Transcriptomic profiling of CFTRhigh and CFTRlow cells showed that the CFTRhigh cells had an elevated inflammatory/innate immune response phenotype. These results encourage caution in interpreting functional data from clonal lines of 16HBE14o- cells, generated after genomic or other manipulations.

Early developmental phenotypes in the cystic fibrosis sheep model.

Highly effective modulator therapies for cystic fibrosis (CF) make it a treatable condition for many people. However, although CF respiratory illness occurs after birth, other organ systems particularly in the digestive tract are damaged before birth. We use an ovine model of CF to investigate the in utero origins of CF disease since the sheep closely mirrors critical aspects of human development. Wildtype (WT) and CFTR -/- sheep tissues were collected at 50, 65, 80, 100, and 120 days of gestation and term (147 days) and used for histological, electrophysiological, and molecular analysis. Histological abnormalities are evident in CFTR-/- -/-  animals by 80 days of gestation, equivalent to 21 weeks in humans. Acinar and ductal dilation, mucus obstruction, and fibrosis are observed in the pancreas; biliary fibrosis, cholestasis, and gallbladder hypoplasia in the liver; and intestinal meconium obstruction, as seen at birth in all large animal models of CF. Concurrently, cystic fibrosis transmembrane conductance regulator (CFTR)-dependent short circuit current is present in WT tracheal epithelium by 80 days gestation and is absent from CFTR -/- tissues. Transcriptomic profiles of tracheal tissues confirm the early expression of CFTR and suggest that its loss does not globally impair tracheal differentiation.

Krüppel-Like Factor 5 Regulates CFTR Expression Through Repression by Maintaining Chromatin Architecture Coupled with Direct Enhancer Activation.

Single cell RNA-sequencing has accurately identified cell types within the human airway that express the Cystic Fibrosis Transmembrane Conductance regulator (CFTR) gene. Low abundance CFTR transcripts are seen in many secretory cells, while high levels are restricted to rare pulmonary ionocytes. Here we focus on the mechanisms coordinating basal CFTR expression in the secretory compartment. Cell-selective regulation of CFTR is achieved within its invariant topologically associating domain by the recruitment of cis-regulatory elements (CREs). CRE activity is coordinated by cell-type-selective transcription factors. One such factor, Krüppel-Like Factor 5 (KLF5), profoundly represses CFTR transcript and protein in primary human airway epithelial cells and airway cell lines. Here we reveal the mechanism of action of KLF5 upon the CFTR gene. We find that depletion or ablation of KLF5 from airway epithelial cells changes higher order chromatin structure at the CFTR locus. Critical looping interactions that are required for normal gene expression are altered, the H3K27ac active chromatin mark is redistributed, and CTCF occupancy is modified. However, mutation of a single KLF5 binding site within a pivotal airway cell CRE abolishes CFTR expression. Hence, KLF5 has both direct activating and indirect repressive effects, which together coordinate CFTR expression in the airway.

Cell function and identity revealed by comparative scRNA-seq analysis in human nasal, bronchial and epididymis epithelia.

The evolutionary relationship of cells within tissues having a similar function but located in different anatomical sites is of considerable biological interest. The development of single-cell RNA sequencing (scRNA-seq) protocols has greatly enhanced opportunities to address this topic. Here we focus on cells in the epithelium which lines two regions of the human respiratory tract and the male genital ducts to delineate the shared, differentiated functions of the different cell populations. Transcriptomic data were used to assess the gene expression profiles of human bronchial, nasal, and epididymal epithelium (HBE, HNE, and HEE). Bulk RNA-seq showed many shared genes expressed in cells from the nasal and bronchial epithelium and highlighted their divergence from the epididymal epithelium. ScRNA-seq in HBE and HNE cells demonstrated overlapping gene expression patterns within basal and secretory cell populations. Moreover, the distribution of cell types was altered in HNE cells derived from donors with cystic fibrosis (CF) when compared to cells from healthy donors. Next, the HBE and HNE datasets were merged and confirmed intersection of cell type gene expression profiles from the two sites. However, secretory and ciliated cells were the most abundant types in the HBE samples, while more basal cells were seen in the HNE populations. We then merged single-cell data from the epididymis to determine if overlapping functions of these cells corresponded to those in the airway. Of note, only the pulmonary ionocytes/epididymis clear cells showed a strongly conserved identity, which was confirmed by imputation in bulk RNA-seq datasets from the same cells.

The Bromodomain Containing 8 (BRD8) transcriptional network in human lung epithelial cells.

Mechanisms regulating gene expression in the airway epithelium underlie its response to the environment. A network of transcription factors (TFs) and architectural proteins, modulate chromatin accessibility and recruit activating or repressive signals. Bromodomain-containing proteins function as TFs or by engaging methyltransferase or acetyltransferase activity to induce chromatin modifications. Here we investigate the role of Bromodomain Containing 8 (BRD8) in coordinating lung epithelial function. Sites of BRD8 occupancy genome-wide were mapped in human lung epithelial cell lines (Calu-3 and 16HBE14o-). CCCTC-Binding Factor (CTCF) was identified as a predicted co-factor of BRD8, based upon motif over-representation under BRD8 ChIP-seq peaks. Following siRNA-mediated depletion of BRD8, differentially expressed genes with nearby peaks of BRD8 occupancy were subject to gene ontology process enrichment analysis. BRD8 targets are enriched for genes involved in the innate immune response and the cell cycle. Depletion of BRD8 increased the secretion of the antimicrobial peptide beta-defensin 1 and multiple chemokines, and reduced cell proliferation.