Activated macrophages undergo a metabolic switch to aerobic glycolysis accumulating Krebs cycle intermediates that alter transcription of immune response genes. Here we extend 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 LPS shock and alleviates familial Mediterranean fever and experimental autoimmune encephalitis (EAE) by targeting GSDMD. Collectively, these findings identify GSDMD as a target of fumarate and reveal a mechanism of action for fumarate-based therapeutics including DMF used to treat multiple sclerosis.

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.

Coronaviruses are a family of RNA viruses that cause acute and chronic diseases of the upper and lower respiratory tract in humans and other animals. SARS-CoV-2 is a recently emerged coronavirus that has led to a global pandemic causing a severe respiratory disease known as COVID-19 with significant morbidity and mortality worldwide. The development of antiviral therapeutics are urgently needed while vaccine programs roll out worldwide. Here we describe a diamidobenzimidazole compound, diABZI-4, that activates STING and is highly effective in limiting SARS-CoV-2 replication in cells and animals. diABZI-4 inhibited SARS-CoV-2 replication in lung epithelial cells. Administration of diABZI-4 intranasally before or even after virus infection conferred complete protection from severe respiratory disease in K18-ACE2-transgenic mice infected with SARS-CoV-2. Intranasal delivery of diABZI-4 induced a rapid short-lived activation of STING, leading to transient proinflammatory cytokine production and lymphocyte activation in the lung associated with inhibition of viral replication. Our study supports the use of diABZI-4 as a host-directed therapy which mobilizes antiviral defenses for the treatment and prevention of COVID-19.

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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-1β. 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.

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.

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 TCRαβ+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.

Recently, a single study revealed a new complex composed of Toll-like receptor 4 (TLR4), TLR6, and CD36 induced by fibrillary Aβ 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 Aβ-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 Aβ. 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.