Gene coexpression networks reveal a broad role for lncRNAs in inflammatory bowel disease.

The role of long noncoding RNAs (lncRNAs) in disease is incompletely understood, but their regulation of inflammation is increasingly appreciated. We addressed the extent of lncRNA involvement in inflammatory bowel disease (IBD) using biopsy-derived RNA-sequencing data from a large cohort of deeply phenotyped patients with IBD. Weighted gene correlation network analysis revealed gene modules of lncRNAs coexpressed with protein-coding genes enriched for biological pathways, correlated with epithelial and immune cell signatures, or correlated with distal colon expression. Correlation of modules with clinical features uncovered a module correlated with disease severity, with an enriched interferon response signature containing the hub lncRNA IRF1-AS1. Connecting genes to IBD-associated single nucleotide polymorphisms (SNPs) revealed an enrichment of SNP-adjacent lncRNAs in biologically relevant modules. Ulcerative colitis-specific SNPs were enriched in distal colon-related modules, suggesting that disease-specific mechanisms may result from altered lncRNA expression. The function of the IBD-associated SNP-adjacent lncRNA IRF1-AS1 was explored in human myeloid cells, and our results suggested IRF1-AS1 promoted optimal production of TNF-α, IL-6, and IL-23. A CRISPR/Cas9-mediated activation screen in THP-1 cells revealed several lncRNAs that modulated LPS-induced TNF-α responses. Overall, this study uncovered the expression patterns of lncRNAs in IBD that identify functional, disease-relevant lncRNAs.

Lnc-ing RNA to intestinal homeostasis and inflammation.

Long noncoding RNAs (lncRNAs) play important roles in numerous biological processes, including the immune system. Initial research in this area focused on cell-based studies, but recent advances underscore the profound significance of lncRNAs at the organismal level, providing invaluable insights into their roles in inflammatory diseases. In this rapidly evolving field, lncRNAs have been described with pivotal roles in the intestinal tract where they regulate intestinal homeostasis and inflammation by influencing processes such as immune cell development, inflammatory signaling pathways, epithelial barrier function, and cellular metabolism. Understanding the regulation and function of lncRNAs in this tissue may position lncRNAs not only as potential disease biomarkers but also as promising targets for therapeutic intervention in inflammatory bowel disease and related diseases.

Targeting STING oligomerization with small-molecule inhibitors.

Stimulator of interferon genes (STING) is an essential adaptor protein required for the inflammatory response to cytosolic DNA. dsDNA activates cGAS to generate cGAMP, which binds and activates STING triggering a conformational change, oligomerization, and the IRF3- and NFκB-dependent transcription of type I Interferons (IFNs) and inflammatory cytokines, as well as the activation of autophagy. Aberrant activation of STING is now linked to a growing number of both rare as well as common chronic inflammatory diseases. Here, we identify and characterize a potent small-molecule inhibitor of STING. This compound, BB-Cl-amidine inhibits STING signaling and production of type I IFNs, IFN-stimulated genes (ISGs) and NFκB-dependent cytokines, but not other pattern recognition receptors. In vivo, BB-Cl-amidine alleviated pathology resulting from accrual of cytosolic DNA in Trex-1 mutant mice. Mechanistically BB-Cl-amidine inhibited STING oligomerization through modification of Cys148. Collectively, our work uncovers an approach to inhibit STING activation and highlights the potential of this strategy for the treatment of STING-driven inflammatory diseases.

The lncRNA HOXA11os regulates mitochondrial function in myeloid cells to maintain intestinal homeostasis.

This study reveals a previously uncharacterized mechanism to restrict intestinal inflammation via a regulatory RNA transcribed from a noncoding genomic locus. We identified a novel transcript of the lncRNA HOXA11os specifically expressed in the distal colon that is reduced to undetectable levels in colitis. HOXA11os is localized to mitochondria under basal conditions and interacts with a core subunit of complex 1 of the electron transport chain (ETC) to maintain its activity. Deficiency of HOXA11os in colonic myeloid cells results in complex I deficiency, dysfunctional oxidative phosphorylation (OXPHOS), and the production of mitochondrial reactive oxygen species (mtROS). As a result, HOXA11os-deficient mice develop spontaneous intestinal inflammation and are hypersusceptible to colitis. Collectively, these studies identify a new regulatory axis whereby a lncRNA maintains intestinal homeostasis and restricts inflammation in the colon through the regulation of complex I activity.

Epithelial HNF4A shapes the intraepithelial lymphocyte compartment via direct regulation of immune signaling molecules.

Hepatocyte nuclear factor 4 α (HNF4A) is a highly conserved nuclear receptor that has been associated with ulcerative colitis. In mice, HNF4A is indispensable for the maintenance of intestinal homeostasis, yet the underlying mechanisms are poorly characterized. Here, we demonstrate that the expression of HNF4A in intestinal epithelial cells (IECs) is required for the proper development and composition of the intraepithelial lymphocyte (IEL) compartment. HNF4A directly regulates expression of immune signaling molecules including butyrophilin-like (Btnl) 1, Btnl6, H2-T3, and Clec2e that control IEC-IEL crosstalk. HNF4A selectively enhances the expansion of natural IELs that are TCRγδ+ or TCRαß+CD8αα+ to shape the composition of IEL compartment. In the small intestine, HNF4A cooperates with its paralog HNF4G, to drive expression of immune signaling molecules. Moreover, the HNF4A-BTNL regulatory axis is conserved in human IECs. Collectively, these findings underscore the importance of HNF4A as a conserved transcription factor controlling IEC-IEL crosstalk and suggest that HNF4A maintains intestinal homeostasis through regulation of the IEL compartment.

Dysbiosis exacerbates colitis by promoting ubiquitination and accumulation of the innate immune adaptor STING in myeloid cells.

Alterations in the cGAS-STING DNA-sensing pathway affect intestinal homeostasis. We sought to delineate the functional role of STING in intestinal inflammation. Increased STING expression was a feature of intestinal inflammation in mice with colitis and in humans afflicted with inflammatory bowel disease. Mice bearing an allele rendering STING constitutively active exhibited spontaneous colitis and dysbiosis, as well as progressive chronic intestinal inflammation and fibrosis. Bone marrow chimera experiments revealed STING accumulation in intestinal macrophages and monocytes as the initial driver of inflammation. Depletion of Gram-negative bacteria prevented STING accumulation in these cells and alleviated intestinal inflammation. STING accumulation occurred at the protein rather than transcript level, suggesting post-translational stabilization. We found that STING was ubiquitinated in myeloid cells, and this K63-linked ubiquitination could be elicited by bacterial products, including cyclic di-GMP. Our findings suggest a positive feedback loop wherein dysbiosis foments the accumulation of STING in intestinal myeloid cells, driving intestinal inflammation.

Succination inactivates gasdermin D and blocks pyroptosis.

Activated macrophages undergo a metabolic switch to aerobic glycolysis, accumulating Krebs' cycle intermediates that alter transcription of immune response genes. We extended these observations by defining fumarate as an inhibitor of pyroptotic cell death. We found that dimethyl fumarate (DMF) delivered to cells or endogenous fumarate reacts with gasdermin D (GSDMD) at critical cysteine residues to form S-(2-succinyl)-cysteine. GSDMD succination prevents its interaction with caspases, limiting its processing, oligomerization, and capacity to induce cell death. In mice, the administration of DMF protects against lipopolysaccharide shock and alleviates familial Mediterranean fever and experimental autoimmune encephalitis by targeting GSDMD. Collectively, these findings identify GSDMD as a target of fumarate and reveal a mechanism of action for fumarate-based therapeutics that include DMF, for the treatment of multiple sclerosis.

TLR2 Dimerization Blockade Allows Generation of Homeostatic Intestinal Macrophages under Acute Colitis Challenge.

Recruited blood monocytes contribute to the establishment, perpetuation, and resolution of tissue inflammation. Specifically, in the inflamed intestine, monocyte ablation was shown to ameliorate colitis scores in preclinical animal models. However, the majority of intestinal macrophages that seed the healthy gut are also monocyte derived. Monocyte ablation aimed to curb inflammation would therefore likely interfere with intestinal homeostasis. In this study, we used a TLR2 trans-membrane peptide that blocks TLR2 dimerization that is critical for TLR2/1 and TLR2/6 heterodimer signaling to blunt inflammation in a murine colitis model. We show that although the TLR2 peptide treatment ameliorated colitis, it allowed recruited monocytes to give rise to macrophages that lack the detrimental proinflammatory gene signature and reduced potentially damaging neutrophil infiltrates. Finally, we demonstrate TLR blocking activity of the peptide on in vitro cultured human monocyte-derived macrophages. Collectively, we provide a significantly improved anti-inflammatory TLR2 peptide and critical insights in its mechanism of action toward future potential use in the clinic.

The HTLV-1 gp21 fusion peptide inhibits antigen specific T-cell activation in-vitro and in mice.

The ability of the Lentivirus HIV-1 to inhibit T-cell activation by its gp41 fusion protein is well documented, yet limited data exists regarding other viral fusion proteins. HIV-1 utilizes membrane binding region of gp41 to inhibit T-cell receptor (TCR) complex activation. Here we examined whether this T-cell suppression strategy is unique to the HIV-1 gp41. We focused on T-cell modulation by the gp21 fusion peptide (FP) of the Human T-lymphotropic Virus 1 (HTLV-1), a Deltaretrovirus that like HIV infects CD4+ T-cells. Using mouse and human in-vitro T-cell models together with in-vivo T-cell hyper activation mouse model, we reveal that HTLV-1's FP inhibits T-cell activation and unlike the HIV FP, bypasses the TCR complex. HTLV FP inhibition induces a decrease in Th1 and an elevation in Th2 responses observed in mRNA, cytokine and transcription factor profiles. Administration of the HTLV FP in a T-cell hyper activation mouse model of multiple sclerosis alleviated symptoms and delayed disease onset. We further pinpointed the modulatory region within HTLV-1's FP to the same region previously identified as the HIV-1 FP active region, suggesting that through convergent evolution both viruses have obtained the ability to modulate T-cells using the same region of their fusion protein. Overall, our findings suggest that fusion protein based T-cell modulation may be a common viral trait.

Intramembrane attenuation of the TLR4-TLR6 dimer impairs receptor assembly and reduces microglia-mediated neurodegeneration.

Recently, a single study revealed a new complex composed of Toll-like receptor 4 (TLR4), TLR6, and CD36 induced by fibrillary Aß peptides, the hallmark of Alzheimer's disease. Unlike TLRs located on the plasma membrane that dimerize on the membrane after ligand binding to their extracellular domain, the TLR4-TLR6-CD36 complex assembly has been suggested to be induced by intracellular signals from CD36, similar to integrin inside-out signaling. However, the assembly site of TLR4-TLR6-CD36 and the domains participating in Aß-induced signaling is still unknown. By interfering with TLR4-TLR6 dimerization using a TLR4-derived peptide, we show that receptor assembly is abrogated within the plasma membrane. Furthermore, we reveal that the transmembrane domains of TLR4 and TLR6 have an essential role in receptor dimerization and activation. Inhibition of TLR4-TLR6 assembly was associated with reduced secretion of proinflammatory mediators from microglia cells, ultimately rescuing neurons from death. Our findings support TLR4-TLR6 dimerization induced by Aß. Moreover, we shed new light on TLR4-TLR6 assembly and localization and show the potential of inhibiting TLR4-TLR6 dimerization as a treatment of Alzheimer's disease.

Neutralization of pro-inflammatory monocytes by targeting TLR2 dimerization ameliorates colitis.

Monocytes have emerged as critical driving force of acute inflammation. Here, we show that inhibition of Toll-like receptor 2(TLR2) dimerization by a TLR2 transmembrane peptide (TLR2-p) ameliorated DSS-induced colitis by interfering specifically with the activation of Ly6C(+) monocytes without affecting their recruitment to the colon. We report that TLR2-p directly interacts with TLR2 within the membrane, leading to inhibition of TLR2-TLR6/1 assembly induced by natural ligands. This was associated with decreased levels of extracellular signal-regulated kinases (ERK) signaling and reduced secretion of pro-inflammatory cytokines, such as interleukin (IL)-6, IL-23, IL-12, and IL-1ß. Altogether, our study provides insights into the essential role of TLR2 dimerization in the activation of pathogenic pro-inflammatory Ly6C(hi) monocytes and suggests that inhibition of this aggregation by TLR2-p might have therapeutic potential in the treatment of acute gut inflammation.

Assembly of the TLR2/6 transmembrane domains is essential for activation and is a target for prevention of sepsis.

TLR2, together with TLR1 and TLR6, is essential for detecting lipopeptides and bacterial cell wall components such as lipoteichoic acid from Gram-positive bacteria. In this study, we report that transmembrane domain (TMD)-derived peptides from TLR2 and TLR6 specifically inhibit TLR2 activation. Secretion of the cytokines TNF-α and IL-6 by cultured macrophages (RAW264.7 cell line) was inhibited by these peptides in response to TLR2 activation by lipoteichoic acid (TLR2/6 activator) or palmitoyl (3)-Cys-Ser-Lys(4)-OH (TLR2/1 activator) but not by LPS (TLR4 activator). Extensive biophysical and biochemical assays, combined with GALLEX experiments, show that these peptides heterodimerize with their complementary TMDs on their reciprocal protein. These results suggest that TLR2/6/1 TMD assembly is essential for activating this complex. Importantly, when administered to mice inflicted by TLR2, but not TLR4-driven lethal inflammation, a selected peptide rescued 60% of these septic mice, showing potent in vivo inhibition of TNF-α and IL-6 secretion. Furthermore, this peptide also showed high protection in a whole bacteria model. Owing to the importance of TLR2 regulation under a variety of pathological conditions, compounds that can fine-tune this activity are of great importance.

D-alanylation of lipoteichoic acids confers resistance to cationic peptides in group B streptococcus by increasing the cell wall density.

Cationic antimicrobial peptides (CAMPs) serve as the first line of defense of the innate immune system against invading microbial pathogens. Gram-positive bacteria can resist CAMPs by modifying their anionic teichoic acids (TAs) with D-alanine, but the exact mechanism of resistance is not fully understood. Here, we utilized various functional and biophysical approaches to investigate the interactions of the human pathogen Group B Streptococcus (GBS) with a series of CAMPs having different properties. The data reveal that: (i) D-alanylation of lipoteichoic acids (LTAs) enhance GBS resistance only to a subset of CAMPs and there is a direct correlation between resistance and CAMPs length and charge density; (ii) resistance due to reduced anionic charge of LTAs is not attributed to decreased amounts of bound peptides to the bacteria; and (iii) D-alanylation most probably alters the conformation of LTAs which results in increasing the cell wall density, as seen by Transmission Electron Microscopy, and reduces the penetration of CAMPs through the cell wall. Furthermore, Atomic Force Microscopy reveals increased surface rigidity of the cell wall of the wild-type GBS strain to more than 20-fold that of the dltA mutant. We propose that D-alanylation of LTAs confers protection against linear CAMPs mainly by decreasing the flexibility and permeability of the cell wall, rather than by reducing the electrostatic interactions of the peptide with the cell surface. Overall, our findings uncover an important protective role of the cell wall against CAMPs and extend our understanding of mechanisms of bacterial resistance.