Noncoding RNA-mediated regulation in innate and adaptive immunity R. Michael Sheetz, PhD Center for Computational Sciences

The immune system and how it works Pathogenic agents represent a constant threat to any organism. In response, mammals (and insects) have evolved a complex network of specialized cells and humoral factors capable of controlling and eliminating these pathogens. The immune system is composed of two key components the innate immune system and the adaptive immune system.

Pathogenic agents represent a constant threat to any organism. In response, mammals (and insects) have evolved a complex network of specialized cells and humoral factors capable of controlling and eliminating these pathogens. The immune system is composed of two key components the innate immune system and the adaptive immune system. Innate immunity provides the host with a first line of defense against infection by inducing both inflammatory responses and antimicrobial pathways. The innate immune system also plays a key role in the initiation and subsequent direction of the adaptive immune system. The immune system and how it works

Pathogenic agents represent a constant threat to any organism. In response, mammals (and insects) have evolved a complex network of specialized cells and humoral factors capable of controlling and eliminating these pathogens. The immune system is composed of two key components the innate immune system and the adaptive immune system. Innate immunity provides the host with a first line of defense against infection by inducing both inflammatory responses and antimicrobial pathways. The innate immune system also plays a key role in the initiation and subsequent direction of the adaptive immune system. Adaptive immunity, which exhibits a delay of 4 to 7 days before its initial response takes effect, evolved to provide a more versatile means of defense as well as increased protection against subsequent re-infection by the same pathogen. Adaptive immunity is induced in response to the presence of a foreign antigen and, in contrast to innate immunity, is mediated by the clonal selection of two classes of antigen-specific WBCs (B-lymphocytes and T-lymphocytes). The immune system and how it works

Key cellular components of the innate immune system Neutrophils PMN cells recruited to the site of infection, phagocytose and kill invading; also contribute to collateral tissue damage that occurs during inflammation Macrophages function in phagocytosis, extracellular killing of infected or altered self target cells, contribute to tissue repair, and act as antigen-presenting cells (APCs) Mast cells associated with wound healing and defense against pathogens; often associated with allergy and anaphylaxis; when activated, release histamine- and heparin- rich granules and chemotactic cytokines into the environment. Basophiles / Eosinophils when activated, basophils release histamine (in defense against parasites), and play a role in allergic reactions (such as asthma) when activated, eosinophils secrete a range of highly toxic proteins and free radicals effective in killing bacteria and parasites (also responsible for tissue damage occurring during allergic reactions) NK (natural killer) cells NK cells nonspecifically kill virus infected and tumor cells.

Dendritic cells DCs main function as antigen presenting cells whose main function is to process antigen material and present it on the cell surface to the T cells of the immune system. DCs serve as a link between the innate and adaptive immune systems. Conventional dendritic cells express the toll-like receptors TLR2 and TLR4 and secrete interleukin 12 (IL 12) Plasmacytoid dendritic cells (pDCs) express the TLRs TLR7 and TLR9 and secrete high amounts of interferon-alpha (IFN ) Key cellular components of the innate immune system Toll-like receptors TLRs represent a type of pattern recognition receptor (PRR) and recognize molecules broadly shared by pathogens collectively referred to as pathogen-associated molecular patterns (PAMPs).

Key cellular components of the adaptive immune system T cells meditate effective immune responses by recognizing antigen through cell-surface T-cell receptors (TCRs). T cells also express one of two cell-surface glycoproteins that are important for TCR signalling, CD4 or CD8. These co-receptor proteins bind invariant regions of MHC class II (CD4) and class I (CD8) molecules, and contribute to TCR-signal transduction through their interactions with membrane-associated signaling molecules. Bosselut, R. Nature Reviews Immunology 4, 529-540 (2004)

Expression of CD4+ T cells are MHC class II restricted and function as T helper (Th) cells when activated, assisting effector components of the immune system (such as B cells) through cytokine secretion and upregulation of expression of specific membrane ligands. By contrast, CD8+ T cells are MHC class I restricted and after activation, acquire cytotoxic properties that allow them to directly kill cells expressing their target antigen. Lazarevic, V. et al. Nature Reviews Immunology 13, 777-789 (2013)

The inflammatory response is the first line of defense against infection and the capacity to repair and restore damaged tissues. If unchecked, a prolonged or inappropriately scaled inflammation can be detrimental to the host and lead to disease such as atherosclerosis, arthritis and cancer. The acute inflammatory response is initiated when pattern recognition receptors (PRRs) present on the surface of phagocytic macrophages recognize and bind common constituents of the surface of a pathogen (e.g., bacterium) triggers a set of signaling cascades that leads to rapid, dynamic and temporally regulated changes in the expression of hundreds of genes involved in antimicrobial defense, phagocytosis, cell migration, tissue repair and the regulation of adaptive immunity.

The inflammatory response is the first line of defense against infection and the capacity to repair and restore damaged tissues. If unchecked, a prolonged or inappropriately scaled inflammation can be detrimental to the host and lead to disease such as atherosclerosis, arthritis and cancer. The acute inflammatory response is initiated when pattern recognition receptors (PRRs) present on the surface of phagocytic macrophages recognize and bind common constituents of the surface of a pathogen (e.g., bacterium) triggers a set of signaling cascades that leads to rapid, dynamic and temporally regulated changes in the expression of hundreds of genes involved in antimicrobial defense, phagocytosis, cell migration, tissue repair and the regulation of adaptive immunity. Phagocytosis of the pathogen induces the macrophage to secrete both cytokines and chemokines Cytokines increase the permeability of blood vessels (allowing fluid and proteins to pass into the tissues) and activate T cells, whereas chemokines direct the migration of neutrophils and monocytes from the bloodstream to the site of infection. The accumulation of fluid and cells at the site of infection causes the redness, swelling, heat, and pain, known collectively as inflammation

This change in gene expression involves multiple layers of regulation that vary depending on the cell lineage involved and the specific signal that is encountered. This regulation of gene expression occurs both at the level of transcription and post-transcription and includes mRNA splicing, mRNA polyadenylation, mRNA stability, and protein translation. The complexity of this regulation allows fine-tuning of both the strength and the duration of the immune response. Within this complexity of immune regulation, non-coding RNAs (both miRNAs and lncRNAs) have emerged as key components in the regulation of both innate and adaptive immunity.

This change in gene expression involves multiple layers of regulation that vary depending on the cell lineage involved and the specific signal that is encountered. This regulation of gene expression occurs both at the level of transcription and post-transcription and includes mRNA splicing, mRNA polyadenylation, mRNA stability, and protein translation. The complexity of this regulation allows fine-tuning of both the strength and the duration of the immune response. Within this complexity of immune regulation, non-coding RNAs (both miRNAs and lncRNAs) have emerged as key components in the regulation of both innate and adaptive immunity. Post-transcriptional regulation in innate and adaptive immunity alternative splicing alternative polyadenylation change in mRNA stability change in translation initiation change in translation elongation

Possible consequences of alternative splicing during pre-mRNA processing Approximately one-fifth of the genes expressed in human dendritic cells (DCs) undergo alternative splicing upon bacterial challenge. Stimulation of human monocytes with the Toll-like receptor 4 (TLR4) ligand LPS and with IFN causes use of proximal poly(A) sites in terminal exons that contain two or more poly(A) sites to be favored. global shortening of 3-UTRs and a loss of key regulatory elements such as miRNA target sites and AU rich elements (AREs) Alternative splicing can alter the sequence of the encoded protein as a result of: mutually exclusive exons exon skipping intron retention alternative use of 5 or 3 splice sites at intron ends Alternative promoter use alternative first exons in length and sequence of the 5-UTR Alternative polyadenylation within an intron mRNA encoding a truncated protein product Alternative polyadenylation within the last exon shortening or extension the 3-UTR

Toll-like receptor (TLR) signaling pathways are regulated through diverse transcripts generated by alternative splicing and alternative polyadenylation Carpenter, S. et al. Nature Reviews Immunol, 14, 361 (2014)

MicroRNA Regulation of the Immune System

Biogenesis and functional mechanisms of miRNAs, piRNAs and snoRNAs Esteller, M. Nature Reviews Genetics 12, 861-874 (2011)

Regulation of miRNA-mediated gene repression by alternative polyadenylation miRNAs regulate gene expression post-transcriptionally by forming imperfect base pairing with sequences in the 3 UTR of mRNAs, thereby reducing protein synthesis by repression of translation or by inducing mRNA destabilization and degradation. Alternative polyadenylation leads to mRNAs with varying length of their 3UTR. Usage of different 3UTR lengths is a way to allow or avoid miRNA-mediated repression. Lymphocyte activation is associated with 3UTR shortening the same gene subject to miRNA- mediated regulation when it has a long 3UTR is less likely to be targeted by miRNAs once it shortens its 3 UTR. Jeker, L.T. and J.A. Bluestone. Immunological Reviews 253, 65-81 (2013)

More than half of the miRNA-binding sites are downstream from the first polyadenylation site, significant level of miRNA regulation may be lost during T-cell proliferation. Genes whose transcript expression increased during T-cell activation more frequently contain miRNA-binding sites exclusively in their extended 3 UTR than genes whose expression decreased, suggesting that they were repressed by miRNAs before activation. Predicted miR-155 and miR-17-92 target genes use the proximal polyadenylation site more frequently than the distal one compared with non-targets in stimulated T cells genes whose expression is needed but that are targeted by the activation- induced miRNAs miR-155 and miR-17-92 may use the short form of their 3UTR to avoid repression during activation, allowing one miRNA to exert differential functions in resting and activated T cells Differential regulation of short versus long 3 UTR usage by predicted miR-155 and miR-17-92 target genes defines a subset of genes for repression while genes that require neutral or induced expression will switch to using the short 3UTR

Schematic representation of an extracellular cue (e.g. cytokine) on a T cell and its consequence on the genetic network in absence and presence of alternative polyadenylation (APA) (A) A cytokine induces a network of genes A-M. (B) miRNA expression allows to shape the gene expression program induced by the cytokine. (C) The interplay of miRNA expression and APA provides a cell additional flexibility to respond to the cytokine. Jeker, L.T. and J.A. Bluestone. Immunological Reviews 253, 65-81 (2013)

Regulation of the TLR4 signaling pathway uses alternative splicing of mRNAs encoding the receptor (TLR4) and the co-receptor (MD2), the adaptor molecules (myeloid differentiation primary response protein 88 (MYD88) and TRIF-related adaptor molecule (TRAM), as well as the IL 1R associated kinases (IRAKs) AP 1, activator protein 1; IRF, interferon-regulatory factor; LPS, lipopolysaccharide; MD2B, splice variant of MD2; MYD88s, splice variant of MYD88; NF B, nuclear factor B; smTLR4, soluble TLR4 splice variant; TAG, splice variant of TRAM; TRAF, TNF receptor-associated factor; TRIF, TIR-domain- containing adaptor protein inducing IFN Carpenter, S. et al. Nature Reviews Immunol, 14, 361 (2014) The use of shorter protein isoforms to fine-tune signaling appears to be a common mechanism that occurs throughout the TLR signaling pathway

T bet (encoded by Tbx21) is an immune cell-specific member of the T box family of transcription factors that functions as a master regulator of commitment to Th1 cell lineage and represents a bridge between innate and adaptive immunity. Together with STAT4, T bet has two central roles in the generation of transcriptionally competent Th1 cell specific genes in CD4+ T cells. T bet-mediates epigenetic changes that are primarily dependent on recruitment of enzymes that generate chromatin modifications associated with either gene activation (histone H3 or H4 acetylation, and H3 lysine 4 (H3K4) dimethylation) or gene repression (H3K27 trimethylation). T bet also organizes the 3D architecture of the Ifng locus by enhancing occupancy of the transcriptional repressor CCCTC-binding factor (CTCF) between the boundaries of the Ifng locus and a +1 kb site. This binding promotes CTCF-dependent chromatin looping, which brings T bet-binding enhancers and CTCF-binding sites in close proximity at the Ifng promoter Ifng expression in TH1 cells. T bet expression in TH1 cells is fine-tuned post-transcriptionally by the microRNA 29 cluster T-bet

Regulation by miRNAs in innate immunity In the immune system, miRNAs have been shown to regulate lineage commitment, proliferation, effector functions, and differentiation in normal and diseased conditions. Activation of the innate immune response with changes in expression of a number of selected miRNAs, including miR-146, miR-155, miR-132, and miR-9. miR-146a is among the most studied miRNAs, and is recognized as a modulator of differentiation and function of cells of both innate and adaptive immunity.

Regulation by miRNAs in innate immunity In the immune system, miRNAs have been shown to regulate lineage commitment, proliferation, effector functions, and differentiation in normal and diseased conditions. Activation of the innate immune response with changes in expression of a number of selected miRNAs, including miR-146, miR-155, miR-132, and miR-9. miR-146a is among the most studied miRNAs, and is recognized as a modulator of differentiation and function of cells of both innate and adaptive immunity. Both miR-155 and miR-146a are important regulators of inflammation. Both are upregulated in response to LPS in monocytes, but show opposing effects, with miR-155 enhancing and miR-146a inhibiting inflammation. miR-146a is expressed throughout the hematopoietic system. Its expression is low in precursors and resting cells (with the exception of Treg cells) but increases with maturation and activation. The role of miR-146a is to act as a brake on the immune system (mice genetically ablated for miR146a exhibit a significant immunoproliferative disorder and premature death.

Regulation by miRNAs in innate immunity In the immune system, miRNAs have been shown to regulate lineage commitment, proliferation, effector functions, and differentiation in normal and diseased conditions. Activation of the innate immune response with changes in expression of a number of selected miRNAs, including miR-146, miR-155, miR-132, and miR-9. miR-146a is among the most studied miRNAs, and is recognized as a modulator of differentiation and function of cells of both innate and adaptive immunity. Both miR-155 and miR-146a are important regulators of inflammation. Both are upregulated in response to LPS in monocytes, but show opposing effects, with miR-155 enhancing and miR-146a inhibiting inflammation. miR-146a is expressed throughout the hematopoietic system. Its expression is low in precursors and resting cells (with the exception of Treg cells) but increases with maturation and activation. The role of miR-146a is to act as a brake on the immune system (mice genetically ablated for miR146a exhibit a significant immunoproliferative disorder and premature death. Aging mice lacking miR-146a spontaneously developed tumors in secondary lymphoid organs, pointing toward an essential role for this miRNA in regulating development and activation of immune cells. Originally identified in a screen for miRNAs induced by LPS stimulation and shown to be a negative regulator of NF- B signaling.

Induction of miR-146a upon stimulation of TLR4 leads to negative regulation of NF- signaling Stimulation of macrophages or mast cells with LPS leads to activation of the NF- B pathway with nuclear translocation of NF- B, where it activates the expression of specific genes, leading to a physiological response. Among the genes transcriptionally induced by NF- B is also pri-miR-146a. NF- B dependent upregulation of miR-146a leads to reduced NF- B activity through direct targeting of IRAK1 and TRAF6. Montagner, S. et al. Immunological Reviews 253, 12-24 (2013)

Involvement of miRNAs in control of T cell development

Activated naive CD4+ T cells were untransduced () or transduced (+) with a lentiviral vector encoding the precursor of miR-125b. The maintenance of high level of miR-125b during naive CD4+ T cells differentiation prevents the acquisition both of the effector memory phenotype (cell membrane expression of the two cytokine receptors IL-2Rb and IL-10Ra) and of effector functions (intracellular accumulation of the cytokines IFN- and IL-13). Enforcement of naive state in primary human lymphocytes by miR-125b

Net importance of miRNAs in control of T cell development T-cell differentiation and peripheral function is shown in schematic form progressing from the thymic stages of double negative (DN), double positive (DP), and CD4 and CD8 single positive (SP) stages, including the tolerance checkpoints of negative selection and regulatory T-cell (Treg) induction. The proportional net importance of the global microRNA network, as measured by the severity of the impact in Dicer-deficient cells, is illustrated along the bottom of the schematic in red.

Regulation by miRNAs in adaptive immunity Expression profiles of microRNA exhibit a dynamic pattern of regulation throughout thymocyte differentiation. Functional analysis of microRNA-deficient thymocytes reveals major differences depending on when the ability to generate mature microRNA is lost. Lck-Cre-mediated excision of Dicer early depletion of the microRNA network 10-fold decline in thymic cellularity, with a defect in differentiation past the DN stage.

Regulation by miRNAs in adaptive immunity Expression profiles of microRNA exhibit a dynamic pattern of regulation throughout thymocyte differentiation. Functional analysis of microRNA-deficient thymocytes reveals major differences depending on when the ability to generate mature microRNA is lost. Lck-Cre-mediated excision of Dicer early depletion of the microRNA network 10-fold decline in thymic cellularity, with a defect in differentiation past the DN stage. Thymocyte differentiation past the DN stage appears to be relatively intact in Lck-Cre-mediated Dicer-deficient mice. Delaying Dicer-excision past the DN-DP transition, Dicer-deficient thymocytes impeded progression from DP to SP stages A major contributor to the function of microRNA during positive selection is thought to be the miR-181 family, which may constitute up to half of the total microRNA content of DP cells.

Regulation by miRNAs in adaptive immunity Expression profiles of microRNA exhibit a dynamic pattern of regulation throughout thymocyte differentiation. Functional analysis of microRNA-deficient thymocytes reveals major differences depending on when the ability to generate mature microRNA is lost. Lck-Cre-mediated excision of Dicer early depletion of the microRNA network 10-fold decline in thymic cellularity, with a defect in differentiation past the DN stage. Thymocyte differentiation past the DN stage appears to be relatively intact in Lck-Cre-mediated Dicer-deficient mice. Delaying Dicer-excision past the DN-DP transition, Dicer-deficient thymocytes impeded progression from DP to SP stages A major contributor to the function of microRNA during positive selection is thought to be the miR-181 family, which may constitute up to half of the total microRNA content of DP cells. The function of elevated miR-181 in thymocytes may be both to aid the positive selection off of weak ligands and to enhance sensitivity to negative selection from strong ligands. miR-181 may have functions in addition to adjusting TCR signal strength, such as modulation of Notch signaling.

miRNA functions in CD4+ T-cell homeostasis, activation, and effector polarization Key cellular events of peripheral CD4+ T cells: maintenance of the naive T-cell pool through homeostasis, proliferation, and activation upon antigen stimulation, and polarization into effector lineages, including IFN -producing Th1 cells, IL-4-producing Th2 cells, and IL-17-producing Th17 cells. The net contribution of the microRNA network to each stage is indicated with either a red plus (indicating a positive effect) or red minus (indicating a negative effect).

miRNA control in the differentiation of CD4+ T helper (Th) cell subsets miRNAs regulate the differentation of different effector (Th1, Th2, Th17, and (Tfh) and regulatory (Treg) subpopulations of CD4+ T helper cells (miRNAs shown in green (red) are reported to positively (negatively) regulate their differentiation). miRNAs regulate the differentation of different effector (Th1, Th2, Th17, and Tfh) and regulatory (Treg) subpopulations of CD4+ T helper cells. miRNAs shown in green (red) are reported to positively (negatively) regulate their differentiation.

Summary of the functions individual miRNAs play in T cells

Long ncRNA Regulation of the Immune System

Studies in macrophages have also revealed important roles for lncRNAs in controlling inflammatory gene expression. Many lncRNAs were found to be dynamically regulated in macrophages that were exposed to TLR2 ligands. One such transcript, lincRNA Cox2, was found to act as a master regulator of gene expression. Regulation by lncRNAs in adaptive immunity

Studies in macrophages have also revealed important roles for lncRNAs in controlling inflammatory gene expression. Many lncRNAs were found to be dynamically regulated in macrophages that were exposed to TLR2 ligands. One such transcript, lincRNA Cox2, was found to act as a master regulator of gene expression. lincRNA Cox2 represses the basal expression of IFN-stimulated genes (ISGs) by partnering with the heterogeneous nuclear ribonucleoproteins (hnRNPs) hnRNPA/B and hnRNPA2/B1. lincRNA Cox2 is also essential for the TLR-induced expression of il6 and more than 700 additional genes (many of which are secondary immune response genes). A pseudogene RNA named Lethe binds RELA (the p65 subunit of NF B heterodimeric complex), which prevents NF B from binding to promoter regions of target genes. In addition, a lincRNA called TNF and hnRNPL-related immunoregulatory lincRNA (THRIL) has been shown to regulate the expression of tumour necrosis factor (TNF) in human monocytes through its interactions with hnRNPL. Regulation by lncRNAs in adaptive immunity

Regulation of inflammatory gene expression by lincRNA-Cox2

The lncRNA Tmevpg1 (a.k.a. NeST) controls Theilers virus persistence in mice by promoting the transcription of Ifng in CD8+ T cells. The Tmevpg1 lncRNA binds to WD repeat-containing protein 5 (WDR5), a histone-modifying complex, altering histone 3 (H3) lysine 4 trimethylation at the Ifng locus.. Regulation by lncRNAs in adaptive immunity

The lncRNA Tmevpg1 (a.k.a. NeST) controls Theilers virus persistence in mice by promoting the transcription of Ifng in CD8+ T cells. The Tmevpg1 lncRNA binds to WD repeat-containing protein 5 (WDR5), a histone-modifying complex, altering histone 3 (H3) lysine 4 trimethylation at the Ifng locus.. lncRNA DQ786243 affects Treg related cAMP response element binding protein (CREB) and Foxp3 expression in Crohns disease. CREB is important for the activity of the TCR response element in the Foxp3 (forkhead box P3, a master transcription factor in function and development of Treg cells). CD4 T cell activation requires low levels of intracellular cAMP, which plays an important role in contact-dependent manner to control the suppressive function of nTreg cells. This finding may provide a interrelationship between a lncRNA (DQ786243 )and a miRNA (miR-142-3p) in immune regulation. Regulation by lncRNAs in adaptive immunity

nTreg contains much higher levels of cAMP than do nave or effector CD4 T cells. nTreg releases intracellular cAMP into effector T cells suppression of effector cell function. Activated helper CD4 T cell contains high levels of PDE, which is down-regulated in the nTreg. Blocking of cAMP degradation by PDE4 inhibitor leads to increased Tregs suppressive function. Elevated levels of intracellular cAMP blocks IL-12 signaling pathway in target cells and thus suppress its differentiation into Th1 lineage. Down-regulation of miR142-3p is essential for Treg function. miR-142-3p maintains low levels of intracellular cAMP by targeting adenylyl cyclase (AC) 9 mRNA. In Treg cells, Foxp3 directly down-regulates miR-142-3p expression and keeps the AC9/cAMP pathway active, elevates intracellular cAMP levels, and down-regulates miR142-3p required for suppressor function of Treg cells. Possible interrelationship between lncRNAs and miRNAs in adaptive immunity

Expression of CD4 or CD8 defines two distinct T-cell lineages that differ both by their MHC specificity and by their function. CD4+ T cells are MHC class II restricted and function as T helper (Th) cells when activated, assisting effector components of the immune system (such as B cells) through cytokine secretion and upregulation of expression of specific membrane ligands. CD8+ T cells are MHC class I restricted and after activation, acquire cytotoxic properties that allow them to directly kill cells expressing their target antigen. Given that CD4+ and CD8+ T cells are derived from a common precursor pool of DP thymocytes, two interesting questions are: Which signals direct thymocytes to either lineage? What are the cytosolic intermediates and nuclear effectors that transform these signals into CD4- or CD8-specific gene-expression programs? It is an intriguing possibility that one or more lncRNAs might be involved in directing this lineage commitment through a mechanism similar to that utilized in XCI, genetic imprinting, or mating-type switching in budding yeast.

Additional references can be provided on ncRNAs, the immune system, autoimmunity, and other immunological disorders if anyone is interested

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Noncoding RNA-mediated regulation in innate and adaptive immunity R. Michael Sheetz, PhD Center for Computational Sciences