Single-cell RNA-seq reveals ectopic and aberrant lung-resident cell populations in idiopathic pulmonary fibrosis.

We provide a single-cell atlas of idiopathic pulmonary fibrosis (IPF), a fatal interstitial lung disease, by profiling 312,928 cells from 32 IPF, 28 smoker and nonsmoker controls, and 18 chronic obstructive pulmonary disease (COPD) lungs. Among epithelial cells enriched in IPF, we identify a previously unidentified population of aberrant basaloid cells that coexpress basal epithelial, mesenchymal, senescence, and developmental markers and are located at the edge of myofibroblast foci in the IPF lung. Among vascular endothelial cells, we identify an ectopically expanded cell population transcriptomically identical to bronchial restricted vascular endothelial cells in IPF. We confirm the presence of both populations by immunohistochemistry and independent datasets. Among stromal cells, we identify IPF myofibroblasts and invasive fibroblasts with partially overlapping cells in control and COPD lungs. Last, we confirm previous findings of profibrotic macrophage populations in the IPF lung. Our comprehensive catalog reveals the complexity and diversity of aberrant cellular populations in IPF.

Limited differential expression of miRNAs and other small RNAs in LPS-stimulated human monocytes.

Monocytes are a distinct subset of myeloid cells with diverse functions in early inflammatory immune modulation. While previous studies have surveyed the role of miRNA regulation on different myeloid cell lines and primary cultures, the time-dependent kinetics of inflammatory stimulation on miRNA expression and the relationship between miRNA-to-target RNA expression have not been comprehensively profiled in monocytes. In this study, we use next-generation sequencing and RT-PCR assays to analyze the non-coding small RNA transcriptome of unstimulated and lipopolysaccharide (LPS)-stimulated monocytes at 6 and 24 hours. We identified a miRNA signature consisting of five mature miRNAs (hsa-mir-146a, hsa-mir-155, hsa-mir-9, hsa-mir-147b, and hsa-mir-193a) upregulated by LPS-stimulated monocytes after 6 hours and found that most miRNAs were also upregulated after 24 hours of stimulation. Only one miRNA gene was down-regulated and no other small RNAs were found dysregulated in monocytes after LPS treatment. In addition, novel tRNA-derived fragments were also discovered in monocytes although none showed significant changes upon LPS stimulation. Interrogation of validated miRNA targets by transcriptomic analysis revealed that absolute expression of most miRNA targets implicating in innate immune response decreased over time in LPS-stimulated monocytes although their expression patterns along the treatment were heterogeneous. Our findings reveal a potential role by which selective miRNA upregulation and stable expression of other small RNAs enable monocytes to develop finely tuned cellular responses during acute inflammation.

Predator-induced phenotypic plasticity of shape and behavior: parallel and unique patterns across sexes and species.

Phenotypic plasticity is often an adaptation of organisms to cope with temporally or spatially heterogenous landscapes. Like other adaptations, one would predict that different species, populations, or sexes might thus show some degree of parallel evolution of plasticity, in the form of parallel reaction norms, when exposed to analogous environmental gradients. Indeed, one might even expect parallelism of plasticity to repeatedly evolve in multiple traits responding to the same gradient, resulting in integrated parallelism of plasticity. In this study, we experimentally tested for parallel patterns of predator-mediated plasticity of size, shape, and behavior of 2 species and sexes of mosquitofish. Examination of behavioral trials indicated that the 2 species showed unique patterns of behavioral plasticity, whereas the 2 sexes in each species showed parallel responses. Fish shape showed parallel patterns of plasticity for both sexes and species, albeit males showed evidence of unique plasticity related to reproductive anatomy. Moreover, patterns of shape plasticity due to predator exposure were broadly parallel to what has been depicted for predator-mediated population divergence in other studies (slender bodies, expanded caudal regions, ventrally located eyes, and reduced male gonopodia). We did not find evidence of phenotypic plasticity in fish size for either species or sex. Hence, our findings support broadly integrated parallelism of plasticity for sexes within species and less integrated parallelism for species. We interpret these findings with respect to their potential broader implications for the interacting roles of adaptation and constraint in the evolutionary origins of parallelism of plasticity in general.

Interleukin-36 potently stimulates human M2 macrophages, Langerhans cells and keratinocytes to produce pro-inflammatory cytokines.

Interleukin (IL)-36 cytokines belong to the IL-1 family and include three agonists, IL-36 α, ß and γ and one inhibitor, IL-36 receptor antagonist (IL-36Ra). IL-36 and IL-1 (α and ß) activate similar intracellular pathways via their related heterodimeric receptors, IL-36R/IL-1RAcP and IL-1R1/IL-1RAcP, respectively. However, excessive IL-36 versus IL-1 signaling induces different phenotypes in humans, which may be related to differential expression of their respective receptors. We examined the expression of IL-36R, IL-1R1 and IL-1RAcP mRNA in human peripheral blood, tonsil and skin immune cells by RT-qPCR. Monocyte-derived dendritic cells (MDDC), M0, M1 or M2-polarized macrophages, primary keratinocytes, dermal macrophages and Langerhans cells (LC) were stimulated with IL-1ß or IL-36ß. Cytokine production was assessed by RT-qPCR and immunoassays. The highest levels of IL-36R mRNA were found in skin-derived keratinocytes, LC, dermal macrophages and dermal CD1a(+) DC. In the blood and in tonsils, IL-36R mRNA was predominantly found in myeloid cells. By contrast, IL-1R1 mRNA was detected in almost all cell types with higher levels in tonsil and skin compared to peripheral blood immune cells. IL-36ß was as potent as IL-1ß in stimulating M2 macrophages, keratinocytes and LC, less potent than IL-1ß in stimulating M0 macrophages and MDDC, and exerted no effects in M1 and dermal macrophages. Levels of IL-1Ra diminished the ability of M2 macrophages to respond to IL-1. Taken together, these data are consistent with the association of excessive IL-36 signaling with an inflammatory skin phenotype and identify human LC and M2 macrophages as new IL-36 target cells.

Sex ratio variation shapes the ecological effects of a globally introduced freshwater fish.

Sex ratio and sexual dimorphism have long been of interest in population and evolutionary ecology, but consequences for communities and ecosystems remain untested. Sex ratio could influence ecological conditions whenever sexual dimorphism is associated with ecological dimorphism in species with strong ecological interactions. We tested for ecological implications of sex ratio variation in the sexually dimorphic western mosquitofish, Gambusia affinis. This species causes strong pelagic trophic cascades and exhibits substantial variation in adult sex ratios. We found that female-biased populations induced stronger pelagic trophic cascades compared with male-biased populations, causing larger changes to key community and ecosystem responses, including zooplankton abundance, phytoplankton abundance, productivity, pH and temperature. The magnitude of such effects indicates that sex ratio is important for mediating the ecological role of mosquitofish. Because both sex ratio variation and sexual dimorphism are common features of natural populations, our findings should encourage broader consideration of the ecological significance of sex ratio variation in nature, including the relative contributions of various sexually dimorphic traits to these effects.

Targeting phosphatase-dependent proteoglycan switch for rheumatoid arthritis therapy.

Despite the availability of several therapies for rheumatoid arthritis (RA) that target the immune system, a large number of RA patients fail to achieve remission. Joint-lining cells, called fibroblast-like synoviocytes (FLS), become activated during RA and mediate joint inflammation and destruction of cartilage and bone. We identify RPTPσ, a transmembrane tyrosine phosphatase, as a therapeutic target for FLS-directed therapy. RPTPσ is reciprocally regulated by interactions with chondroitin sulfate or heparan sulfate containing extracellular proteoglycans in a mechanism called the proteoglycan switch. We show that the proteoglycan switch regulates FLS function. Incubation of FLS with a proteoglycan-binding RPTPσ decoy protein inhibited cell invasiveness and attachment to cartilage by disrupting a constitutive interaction between RPTPσ and the heparan sulfate proteoglycan syndecan-4. RPTPσ mediated the effect of proteoglycans on FLS signaling by regulating the phosphorylation and cytoskeletal localization of ezrin. Furthermore, administration of the RPTPσ decoy protein ameliorated in vivo human FLS invasiveness and arthritis severity in the K/BxN serum transfer model of RA. Our data demonstrate that FLS are regulated by an RPTPσ-dependent proteoglycan switch in vivo, which can be targeted for RA therapy. We envision that therapies targeting the proteoglycan switch or its intracellular pathway in FLS could be effective as a monotherapy or in combination with currently available immune-targeted agents to improve control of disease activity in RA patients.

Immune modulation by butyrophilins.

The B7 family of co-stimulatory molecules has an important role in driving the activation and inhibition of immune cells. Evolving data have shown that a related family of molecules - the butyrophilins - have similar immunomodulatory functions to B7 family members and may represent a novel subset of co-stimulatory molecules. These studies have taken the field by surprise, as the butyrophilins were previously thought to only be important in lactation and milk production. In this Review, we describe the expression patterns of the various members of the butyrophilin family and explore their immunomodulatory functions. In particular, we emphasize the contribution of butyrophilins to immune homeostasis and discuss the potential of targeting these molecules for therapeutic purposes.

Butyrophilin-like 2 modulates B7 costimulation to induce Foxp3 expression and regulatory T cell development in mature T cells.

Naive T cell activation involves at least two signals from an APC, one through the TCR via interaction with peptide-MHC complexes and a second through ligation of CD28 with B7 ligands. Following activation, T cells upregulate a host of other membrane-bound costimulatory molecules that can either promote or inhibit further T cell maturation and proliferation. In some cases, it is necessary to attenuate T cell activation to prevent deleterious inflammation, and inhibitory members of the B7/butyrophilin family of ligands have evolved to balance the strong stimuli the activating B7 ligands confer. Human genetic association and in vitro studies have implicated one such ligand, BTNL2, in controlling inflammation at mucosal surfaces. In this study, we show that recombinant mouse BTNL2 modifies B7/CD28 signaling to promote expression of Foxp3, a transcription factor necessary for regulatory T cell (Treg) development and function. BTNL2 blocks Akt-mediated inactivation of Foxo1, a transcription factor necessary for Foxp3 expression. Immunophenotyping and gene profiling reveal that BTNL2-induced Treg share many properties with natural Treg, and in vivo they suppress enteritis induced by mouse effector T cells. These findings describe a mechanism by which environmental Ag-specific Tregs may be induced by APC expressing specific modulators of costimulatory signals.

Protein tyrosine phosphatase expression profile of rheumatoid arthritis fibroblast-like synoviocytes: a novel role of SH2 domain-containing phosphatase 2 as a modulator of invasion and survival.

OBJECTIVE: The fibroblast-like synoviocytes (FLS) in the synovial intimal lining of the joint are key mediators of inflammation and joint destruction in rheumatoid arthritis (RA). In RA, these cells aggressively invade the extracellular matrix, producing cartilage-degrading proteases and inflammatory cytokines. The behavior of FLS is controlled by multiple interconnected signal transduction pathways involving reversible phosphorylation of proteins on tyrosine residues. However, little is known about the role of the protein tyrosine phosphatases (PTPs) in FLS function. This study was undertaken to explore the expression of all of the PTP genes (the PTPome) in FLS. METHODS: A comparative screening of the expression of the PTPome in FLS from patients with RA and patients with osteoarthritis (OA) was conducted. The functional effect on RA FLS of SH2 domain-containing phosphatase 2 (SHP-2), a PTP that was up-regulated in RA, was then analyzed by knockdown using cell-permeable antisense oligonucleotides. RESULTS: PTPN11 was overexpressed in RA FLS compared to OA FLS. Knockdown of PTPN11, which encodes SHP-2, reduced the invasion, migration, adhesion, spreading, and survival of RA FLS. Additionally, signaling in response to growth factors and inflammatory cytokines was impaired by SHP-2 knockdown. RA FLS that were deficient in SHP-2 exhibited decreased activation of focal adhesion kinase and mitogen-activated protein kinases. CONCLUSION: These findings indicate that SHP-2 has a novel role in mediating human FLS function and suggest that it promotes the invasiveness and survival of RA FLS. Further investigation may reveal SHP-2 to be a candidate therapeutic target for RA.

Optimization of a Potent, Orally Active S1P1 Agonist Containing a Quinolinone Core.

The optimization of a series of S1P1 agonists with limited activity against S1P3 is reported. A polar headgroup was used to improve the physicochemical and pharmacokinetic parameters of lead quinolinone 6. When dosed orally at 1 and 3 mg/kg, the azahydroxymethyl analogue 22 achieved statistically significant lowering of circulating blood lymphocytes 24 h postdose. In rats, a dose-proportional increase in exposure was measured when 22 was dosed orally at 2 and 100 mg/kg.

Quinolinone-based agonists of S1P1: use of a N-scan SAR strategy to optimize in vitro and in vivo activity.

We reveal how a N-scan SAR strategy (systematic substitution of each CH group with a N atom) was employed for quinolinone-based S1P(1) agonist 5 to modulate physicochemical properties and optimize in vitro and in vivo activity. The diaza-analog 17 displays improved potency (hS1P(1) RI; 17: EC(50)=0.020 µM, 120% efficacy; 5: EC(50)=0.070 µM, 110% efficacy) and selectivity (hS1P(3) Ca(2+) flux; 17: EC(50) >25 µM; 5: EC(50)=1.5 µM, 92% efficacy), as well as enhanced pharmacokinetics (17: CL=0.15 L/h/kg, V(dss)=5.1L/kg, T(1/2)=24h, %F=110; 5: CL=0.93L/h/kg, V(dss)=11L/kg, T(1/2)=15 h, %F=60) and pharmacodynamics (17: 1.0mg/kg po, 24h PLC POC=-67%; 5: 3mg/kg po, 24h PLC POC=-51%) in rat.

Loss of the receptor tyrosine kinase Axl leads to enhanced inflammation in the CNS and delayed removal of myelin debris during experimental autoimmune encephalomyelitis.

BACKGROUND: Axl, together with Tyro3 and Mer, constitute the TAM family of receptor tyrosine kinases. In the nervous system, Axl and its ligand Growth-arrest-specific protein 6 (Gas6) are expressed on multiple cell types. Axl functions in dampening the immune response, regulating cytokine secretion, clearing apoptotic cells and debris, and maintaining cell survival. Axl is upregulated in various disease states, such as in the cuprizone toxicity-induced model of demyelination and in multiple sclerosis (MS) lesions, suggesting that it plays a role in disease pathogenesis. To test for this, we studied the susceptibility of Axl-/- mice to experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. METHODS: WT and Axl-/- mice were immunized with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide emulsified in complete Freund's adjuvant and injected with pertussis toxin on day 0 and day 2. Mice were monitored daily for clinical signs of disease and analyzed for pathology during the acute phase of disease. Immunological responses were monitored by flow cytometry, cytokine analysis and proliferation assays. RESULTS: Axl-/- mice had a significantly more severe acute phase of EAE than WT mice. Axl-/- mice had more spinal cord lesions with larger inflammatory cuffs, more demyelination, and more axonal damage than WT mice during EAE. Strikingly, lesions in Axl-/- mice had more intense Oil-Red-O staining indicative of inefficient clearance of myelin debris. Fewer activated microglia/macrophages (Iba1+) were found in and/or surrounding lesions in Axl-/- mice relative to WT mice. In contrast, no significant differences were noted in immune cell responses between naïve and sensitized animals. CONCLUSIONS: These data show that Axl alleviates EAE disease progression and suggests that in EAE Axl functions in the recruitment of microglia/macrophages and in the clearance of debris following demyelination. In addition, these data provide further support that administration of the Axl ligand Gas6 could be therapeutic for immune-mediated demyelinating diseases.

4-Methoxy-N-[2-(trifluoromethyl)biphenyl-4-ylcarbamoyl]nicotinamide: A Potent and Selective Agonist of S1P1.

The sphingosine-1-phosphate-1 receptor (S1P1) and its endogenous ligand sphingosine-1-phosphate (S1P) cooperatively regulate lymphocyte trafficking from the lymphatic system. Herein, we disclose 4-methoxy-N-[2-(trifluoromethyl)biphenyl-4-ylcarbamoyl]nicotinamide (8), an uncommon example of a synthetic S1P1 agonist lacking a polar headgroup, which is shown to effect dramatic reduction of circulating lymphocytes (POC = -78%) in rat 24 h after a single oral dose (1 mg/kg). The excellent potency that 8 exhibits toward S1P1 (EC50 = 0.035 µM, 96% efficacy) and the >100-fold selectivity that it displays against receptor subtypes S1P2-5 suggest that it may serve as a valuable tool to understand the clinical relevance of selective S1P1 agonism.

Discovery of AMG 369, a Thiazolo[5,4-b]pyridine Agonist of S1P1 and S1P5.

The optimization of a series of thiazolopyridine S1P1 agonists with limited activity at the S1P3 receptor is reported. These efforts resulted in the discovery of 1-(3-fluoro-4-(5-(1-phenylcyclopropyl)thiazolo-[5,4-b]pyridin-2-yl)benzyl)azetidine-3-carboxylic acid (5d, AMG 369), a potent dual S1P1/S1P5 agonist with limited activity at S1P3 and no activity at S1P2/S1P4. Dosed orally at 0.1 mg/kg, 5d is shown to reduce blood lymphocyte counts 24 h postdose and delay the onset and reduce the severity of experimental autoimmune encephalomyelitis in rat.

Diverse functions of reactive cysteines facilitate unique biosynthetic processes of aggregate-prone interleukin-31.

Interleukin-31 (IL-31) is a member of the four helical-bundle gp130/IL-6 cytokine family. Despite its implicated roles in inflammatory diseases, the biosynthetic processes of IL-31 have been poorly investigated. A detailed understanding of IL-31 biosynthesis and the nature of ligand-receptor interactions can provide insights into effective strategies for the design of therapeutic approaches. By using various heterologous protein expression systems, we demonstrated that murine IL-31 was secreted as inter-molecularly disulfide-bonded covalent aggregates. Covalently aggregated IL-31 appeared while trafficking in the secretory pathway, but was not actively retained in the ER. The aggregate formation was not caused by a dysfunctional ER quality control mechanism or an intrinsic limitation in protein folding capacity. Furthermore, secreted IL-31 aggregates were part of a large complex composed of various pleiotropic secretory factors and immune-stimulators. The extent and the heterogeneous nature of aggregates may imply that IL-31 was erroneously folded, but it was capable of signaling through cognate receptors. Mutagenesis revealed the promiscuity of all five cysteines in inter-molecular disulfide formation with components of the hetero-aggregates, but no cysteine was required for IL-31 secretion itself. Our present study not only illustrated various functions that cysteines perform during IL-31 biosynthesis and secretion, but also highlighted their potential roles in cytokine effector functions.

Melting Alpine glaciers enrich high-elevation lakes with reactive nitrogen.

Alpine glaciers have receded substantially over the last century in many regions of the world. Resulting changes in glacial runoff not only affect the hydrological cycle, but can also alter the physical (i.e., turbidity from glacial flour) and biogeochemical properties of downstream ecosystems. Here we compare nutrient concentrations, transparency gradients, algal biomass, and fossil diatom species richness in two sets of high-elevation lakes: those fed by snowpack melt alone (SF lakes) and those fed by both glacial and snowpack meltwaters (GSF lakes). We found that nitrate (NO(3)(-)) concentrations in the GSF lakes were 1-2 orders of magnitude higher than in SF lakes. Although nitrogen (N) limitation is common in alpine lakes, algal biomass was lower in highly N-enriched GSF lakes than in the N-poor SF lakes. Contrary to expectations, GSF lakes were more transparent than SF lakes to ultraviolet and equally transparent to photosynthetically active radiation. Sediment diatom assemblages had lower taxonomic richness in the GSF lakes, a feature that has persisted over the last century. Our results demonstrate that the presence of glaciers on alpine watersheds more strongly influences NO(3)(-)concentrations in high-elevation lake ecosystems than any other geomorphic or biogeographic characteristic.

Gatekeepers of intestinal inflammation.

The intestine is subjected to a barrage of insults from food, bacterial flora, and pathogens. Despite this constant antigenic challenge, the mucosal tissues lining the intestinal tract remain largely under control. The mechanisms regulating the homeostatic balance in the gut have been investigated for many years by many groups, but the precise nature of the regulatory control remains elusive. In this review, we provide an overview of pathways proposed to be involved in dampening the inflammatory response and maintaining the homeostatic balance in the intestine, and how these pathways may be disrupted in ulcerative colitis and Crohn's disease.

The oligodendrocyte-specific G protein-coupled receptor GPR17 is a cell-intrinsic timer of myelination.

The basic helix-loop-helix transcription factor Olig1 promotes oligodendrocyte maturation and is required for myelin repair. We characterized an Olig1-regulated G protein-coupled receptor, GPR17, whose function is to oppose the action of Olig1. Gpr17 was restricted to oligodendrocyte lineage cells, but was downregulated during the peak period of myelination and in adulthood. Transgenic mice with sustained Gpr17 expression in oligodendrocytes exhibited stereotypic features of myelinating disorders in the CNS. Gpr17 overexpression inhibited oligodendrocyte differentiation and maturation both in vivo and in vitro. Conversely, Gpr17 knockout mice showed early onset of oligodendrocyte myelination. The opposing action of Gpr17 on oligodendrocyte maturation reflects, at least partially, upregulation and nuclear translocation of the potent oligodendrocyte differentiation inhibitors ID2/4. Collectively, these findings suggest that GPR17 orchestrates the transition between immature and myelinating oligodendrocytes via an ID protein-mediated negative regulation and may serve as a potential therapeutic target for CNS myelin repair.

Recent developments in IBD and the B7 family of costimulatory molecules.

The cells and tissues of the intestinal tract are subjected to a constant onslaught of antigenic challenge from both beneficial and harmful pathogens. Despite this constant stimulation, the host is able to maintain a relatively stable environment, often referred to as 'a controlled state of inflammation'. In patients with chronic inflammatory bowel disease, this controlled state of inflammation is lost. The cause of this loss of control is not fully understood, but there is emerging research interest in positive and negative costimulatory pathways as potential targets for modulating the dysregulation. This review describes the B7 and B7-like butyrophilin families of costimulatory molecules, with an emphasis on the role of costimulation in intestinal inflammation.

Regulation of costimulation in the era of butyrophilins.

The butyrophilin and butyrophilin-like superfamily of molecules has garnered attention in the immunology world in the past few years as a result of the observation that the butyrophilin-like 2 molecule, BTNL2, can alter T cell responsiveness. Additional interest in this superfamily solidified following the discovery that genetic polymorphisms in BTNL2 are associated with predisposition to many human diseases. In this review, we will provide an overview of the members comprising the butyrophilin superfamily of molecules. We will then discuss BTNL2 immunomodulatory function, and BTNL2 structural associations with other costimulatory molecules. We will then draw your attention to some of the lesser-known butyrophilin superfamily members by describing the expression patterns of these molecules in human tissues and cells. And we will finish by hypothesizing on the potential influence on general immune homeostasis that might be mediated by this, thus-far little-studied, family of molecules.