CISH attenuates homeostatic cytokine signaling to promote lung-specific macrophage programming and function.

Tissue-specific cytokine stimuli orchestrate specialized homeostatic functions of resident macrophages. In the lung, steady-state signaling by the cytokine GM-CSF is critical for alveolar macrophage (AM) development and function. Here, we showed that CISH, a suppressor of cytokine signaling (SOCS) family member that is acutely induced by diverse cytokine stimuli in many tissues, was expressed constitutively in AMs in response to steady-state GM-CSF signaling. Cish deficiency led to the generation of foamy AMs and the accumulation of pulmonary surfactant. These phenotypic changes were associated with enhanced activation of STAT5, AKT, and ERK and increased expression of the gene encoding the transcription factor GATA2. RNA-seq analysis of Cish−/− AMs revealed a set of dysregulated immune and lipid-process modules, including the increased expression of genes enriched for GATA2-binding motifs. Last, Cish-deficient, bone marrow­derived macrophages showed increased Gata2 expression and accumulated more lipid upon incubation with bronchoalveolar lavage fluid compared with Cish-sufficient cells. Thus, CISH is part of a feedback loop that constrains homeostatic cytokine signaling and Gata2 expression to maintain AM identity and function.

Nonpeptidergic neurons suppress mast cells via glutamate to maintain skin homeostasis.

Cutaneous mast cells mediate numerous skin inflammatory processes and have anatomical and functional associations with sensory afferent neurons. We reveal that epidermal nerve endings from a subset of sensory nonpeptidergic neurons expressing MrgprD are reduced by the absence of Langerhans cells. Loss of epidermal innervation or ablation of MrgprD-expressing neurons increased expression of a mast cell gene module, including the activating receptor, Mrgprb2, resulting in increased mast cell degranulation and cutaneous inflammation in multiple disease models. Agonism of MrgprD-expressing neurons reduced expression of module genes and suppressed mast cell responses. MrgprD-expressing neurons released glutamate which was increased by MrgprD agonism. Inhibiting glutamate release or glutamate receptor binding yielded hyperresponsive mast cells with a genomic state similar to that in mice lacking MrgprD-expressing neurons. These data demonstrate that MrgprD-expressing neurons suppress mast cell hyperresponsiveness and skin inflammation via glutamate release, thereby revealing an unexpected neuroimmune mechanism maintaining cutaneous immune homeostasis.

Competition for Active TGFß Cytokine Allows for Selective Retention of Antigen-Specific Tissue- Resident Memory T Cells in the Epidermal Niche.

Following antigen-driven expansion in lymph node, transforming growth factor-ß (TGFß) is required for differentiation of skin-recruited CD8+ T cell effectors into epidermal resident memory T (Trm) cells and their epidermal persistence. We found that the source of TGFß -supporting Trm cells was autocrine. In addition, antigen-specific Trm cells that encountered cognate antigen in the skin, and bystander Trm cells that did not, both displayed long-term persistence in the epidermis under steady-state conditions. However, when the active-TGFß was limited or when new T cell clones were recruited into the epidermis, antigen-specific Trm cells were more efficiently retained than bystander Trm cells. Genetically enforced TGFßR signaling allowed bystander Trm cells to persist in the epidermis as efficiently as antigen-specific Trm cells in both contexts. Thus, competition between T cells for active TGFß represents an unappreciated selective pressure that promotes the accumulation and persistence of antigen-specific Trm cells in the epidermal niche.

Genomic programming of IRF4-expressing human Langerhans cells.

Langerhans cells (LC) can prime tolerogenic as well as immunogenic responses in skin, but the genomic states and transcription factors (TF) regulating these context-specific responses are unclear. Bulk and single-cell transcriptional profiling demonstrates that human migratory LCs are robustly programmed for MHC-I and MHC-II antigen presentation. Chromatin analysis reveals enrichment of ETS-IRF and AP1-IRF composite regulatory elements in antigen-presentation genes, coinciding with expression of the TFs, PU.1, IRF4 and BATF3 but not IRF8. Migration of LCs from the epidermis is accompanied by upregulation of IRF4, antigen processing components and co-stimulatory molecules. TNF stimulation augments LC cross-presentation while attenuating IRF4 expression. CRISPR-mediated editing reveals IRF4 to positively regulate the LC activation programme, but repress NF2EL2 and NF-kB pathway genes that promote responsiveness to oxidative stress and inflammatory cytokines. Thus, IRF4-dependent genomic programming of human migratory LCs appears to enable LC maturation while attenuating excessive inflammatory and immunogenic responses in the epidermis.

Charting the cis-regulome of activated B cells by coupling structural and functional genomics.

Cis-regulomes underlying immune-cell-specific genomic states have been extensively analyzed by structure-based chromatin profiling. By coupling such approaches with a high-throughput enhancer screen (self-transcribing active regulatory region sequencing (STARR-seq)), we assembled a functional cis-regulome for lipopolysaccharide-activated B cells. Functional enhancers, in contrast with accessible chromatin regions that lack enhancer activity, were enriched for enhancer RNAs (eRNAs) and preferentially interacted in vivo with B cell lineage-determining transcription factors. Interestingly, preferential combinatorial binding by these transcription factors was not associated with differential enrichment of their sites. Instead, active enhancers were resolved by principal component analysis (PCA) from all accessible regions by co-varying transcription factor motif scores involving a distinct set of signaling-induced transcription factors. High-resolution chromosome conformation capture (Hi-C) analysis revealed multiplex, activated enhancer-promoter configurations encompassing numerous multi-enhancer genes and multi-genic enhancers engaged in the control of divergent molecular pathways. Motif analysis of pathway-specific enhancers provides a catalog of diverse transcription factor codes for biological processes encompassing B cell activation, cycling and differentiation.

Author Correction: Single-cell analysis of mixed-lineage states leading to a binary cell fate choice.

In this Letter, the first name of author Virendra K. Chaudhri was incorrectly spelled 'Viren'; author Meenakshi Venkatasubramanian should also be associated with 'Department of Electrical Engineering and Computer Science, University of Cincinnati, Cincinnati, Ohio 45221, USA'; authors Bruce J. Aronow, Nathan Salomonis, Harinder Singh and H. Leighton Grimes should also be associated with 'Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45229, USA'. The Letter has not been corrected online.

Regulation of bifurcating B cell trajectories by mutual antagonism between transcription factors IRF4 and IRF8.

Upon recognition of antigen, B cells undertake a bifurcated response in which some cells rapidly differentiate into plasmablasts while others undergo affinity maturation in germinal centers (GCs). Here we identified a double-negative feedback loop between the transcription factors IRF4 and IRF8 that regulated the initial developmental bifurcation of activated B cells as well as the GC response. IRF8 dampened signaling via the B cell antigen receptor (BCR), facilitated antigen-specific interaction with helper T cells, and promoted antibody affinity maturation while antagonizing IRF4-driven differentiation of plasmablasts. Genomic analysis revealed concentration-dependent actions of IRF4 and IRF8 in regulating distinct gene-expression programs. Stochastic modeling suggested that the double-negative feedback was sufficient to initiate bifurcation of the B cell developmental trajectories.

Metabolic alterations in lung cancer-associated fibroblasts correlated with increased glycolytic metabolism of the tumor.

Cancer cells undergo a metabolic reprogramming but little is known about metabolic alterations of other cells within tumors. We use mass spectrometry-based profiling and a metabolic pathway-based systems analysis to compare 21 primary human lung cancer-associated fibroblast lines (CAF) to "normal" fibroblast lines (NF) generated from adjacent nonneoplastic lung tissue. CAFs are protumorigenic, although the mechanisms by which CAFs support tumors have not been elucidated. We have identified several pathways whose metabolite abundance globally distinguished CAFs from NFs, suggesting that metabolic alterations are not limited to cancer cells. In addition, we found metabolic differences between CAFs from high and low glycolytic tumors that might reflect distinct roles of CAFs related to the tumor's glycolytic capacity. One such change was an increase of dipeptides in CAFs. Dipeptides primarily arise from the breakdown of proteins. We found in CAFs an increase in basal macroautophagy which likely accounts for the increase in dipeptides. Furthermore, we show a difference between CAFs and NFs in the induction of autophagy promoted by reduced glucose. In sum, our data suggest that increased autophagy may account for metabolic differences between CAFs and NFs and may play additional as yet undetermined roles in lung cancer.

Regulatory cascades of protein phosphatases: implications for cancer treatment.

Coordinated coupling of biochemical reactions involving protein phosphorylation and dephosphorylation represents the hallmark of the intracellular signal transduction machinery. Distinct classes of enzymes known as kinases and phosphatases respectively drive these reactions. Alterations in activity of such signaling intermediates, either due to mutations in the corresponding genes or epigenetic modulation of their expression levels, is often the cause of many cancers. The role of kinases during signal transduction has been extensively investigated over the past several decades and the consensus view is that subsets of kinases form distinct cascades of signaling pathways. Further, the extensive crosstalk that exists between these cascades leads to a complex network configuration for the signaling machinery. Inhibitors of many of these kinases are now being exploited in cancer therapy. In contrast to this, regulation by cellular phosphatases has generally been considered to occur through isolated interactions between a given phosphatase and its target substrate. Emerging evidence, however, is beginning to suggest that phosphatases also inter-regulate each other, and that such interactions can lead to the formation of discrete phosphatase-specific cascades. A phosphatase cascade may be defined broadly as a series of successive dephosphorylation reactions that occur within a cell and are catalyzed by phosphatases which are activated sequentially. In general, the term phosphatase cascade refers to cascades that include two or more phosphatase members. The crosstalk between such regulatory axes of phosphatases and kinase cascades provides for complex modes of regulation, with non-linear signal input/output relationships. This review discusses the implications of such phosphatase-constituted regulatory elements for both signal processing and transmission. Further, we also explore the potential that insights on the functioning of phosphatase cascades offers, for the development of new and selective strategies for cancer therapy.

Integration of a phosphatase cascade with the mitogen-activated protein kinase pathway provides for a novel signal processing function.

We mathematically modeled the receptor-dependent mitogen-activated protein kinase (MAPK) signaling by incorporating the regulation through cellular phosphatases. Activation induced the alignment of a phosphatase cascade in parallel with the MAPK pathway. A novel regulatory motif was, thus, generated, providing for the combinatorial control of each MAPK intermediate. This ensured a non-linear mode of signal transmission with the output being shaped by the balance between the strength of input signal and the activity gradient along the phosphatase axis. Shifts in this balance yielded modulations in topology of the motif, thereby expanding the repertoire of output responses. Thus, we identify an added dimension to signal processing wherein the output response to an external stimulus is additionally filtered through indicators that define the phenotypic status of the cell.