Flavanols and Flavanes from Crinum asiaticum and Their Effects on LPS Signaling Pathway Through the Inhibition of NF-κB Activation

Three new flavanols, (2R,3S)-7-methoxy-flavan-3-ol (1), (2R,3S)-7-hydroxy-flavan-3-ol (2), and (2R,3S)-2′-hydroxy-7-methoxy-flavan-3-ol (3), together with two known flavans (4 and 5), were isolated from the chloroform extract of Crinum asiaticum. Their structures were elucidated by various spectroscopic methods, including 1D and 2D NMR, HR-ESI-MS, and CD data. The isolated compounds 1 and 3−5 showed inhibitory activity toward LPS-induced nitric oxide (NO) production. Further investigation of the NF-κB pathway mechanisms indicated that 1 and 3−5 inhibited the LPS-induced IL-6 production and p65 subunit phosphorylation of NF-κB in RAW264.7 cells, with an effective dose of 10 µM.

Intracellular and Extracellular Lipopolysaccharide Signaling in Sepsis: Avenues for Novel Therapeutic Strategies

Sepsis is defined as organ dysfunction due to a dysregulated systemic host response to infection. During gram-negative bacterial infection and other acute illness such as absorption from the gut infection, lipopolysaccharide (LPS) is a major mediator in sepsis. LPS is able to trigger inflammation through both intracellular and extracellular pathways. Classical interactions between LPS and host cells first involve LPS binding to LPS binding protein (LBP), a carrier. The LPS-LBP complex then binds to a receptor complex including the CD14, MD2, and toll-like receptor 4 (TLR4) proteins, initiating a signal cascade which triggers the secretion of pro-inflammatory cytokines. However, it has been established that LPS is also internalized by macrophages and endothelial cells through TLR4-independent pathways. Once internalized, LPS is able to bind to the cytosolic receptors caspases-4/5 in humans and the homologous caspase-11 in mice. Bound caspases-4/5 oligomerize and trigger the assembly of the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 inflammasome followed by the activation of inflammatory caspase-1 resulting in subsequent release of interleukin-1β. Caspases-4/5 also activate the perforin gasdermin D and purinergic receptor P2X7, inducing cell lysis and pyroptosis. Pyroptosis is a notable source of inflammation and damage to the lung endothelial barrier during sepsis. Thus, inhibition of caspases-4/5/1 or downstream effectors to block intracellular LPS signaling may be a promising therapeutic approach in adjunction with neutralizing extracellular LPS for treatment of sepsis.

Early immune innate hallmarks and microbiome changes across the gut during Escherichia coli O157: H7 infection in cattle

The zoonotic enterohemorrhagic Escherichia coli (EHEC) O157: H7 bacterium causes diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS) in humans. Cattle are primary reservoirs and EHEC O157: H7; the bacteria predominately inhabit the colon and recto-anal junctions (RAJ). The early innate immune reactions in the infected gut are critical in the pathogenesis of EHEC O157: H7. In this study, calves orally inoculated with EHEC O157: H7 showed infiltration of neutrophils in the lamina propria of ileum and RAJ at 7 and 14 days post-infection. Infected calves had altered mucin layer and mast cell populations across small and large intestines. There were differential transcription expressions of key bovine β defensins, tracheal antimicrobial peptide (TAP) in the ileum, and lingual antimicrobial peptide (LAP) in RAJ. The main Gram-negative bacterial/LPS signaling Toll-Like receptor 4 (TLR4) was downregulated in RAJ. Intestinal infection with EHEC O157: H7 impacted the gut bacterial communities and influenced the relative abundance of Negativibacillus and Erysipelotrichaceae in mucosa-associated bacteria in the rectum. Thus, innate immunity in the gut of calves showed unique characteristics during infection with EHEC O157: H7, which occurred in the absence of major clinical manifestations but denoted an active immunological niche.

Resolution-Associated Lactoferrin Peptides Limit LPS Signaling and Cytokine Secretion from Human Macrophages

The neutrophil granule protein lactoferrin is cleaved and accumulates in efferocytic macrophages as inflammation is resolved. Two peptides present within a resolution-associated 17 kDa fragment of lactoferrin promote the termination of inflammation in vivo by enhancing murine macrophage reprogramming. Here, we report that these two bioactive tripeptides, phenylalanine-lysine-aspartic acid and phenylalanine-lysine-glutamic acid (FKD and FKE, respectively), inhibit ERK and cJun activation following human macrophage exposure to LPS. In addition, these peptides at low concentrations (1–10 μM) modulate human macrophage reprogramming to an anti-inflammatory/pro-resolving phenotype. This was reflected by inhibition of LPS-induced TNF-α and IL-6 secretion and increased IL-10 levels. Moreover, we found naturally occurring FKE analogs (FKECH and FKECHLA) can recapitulate the activity of the short peptide in regulating macrophage cytokine secretion, whereas a reversed EKF peptide was inert in this respect. Curiously, FKD and FKE also regulated cytokine production by bone marrow-derived mouse macrophages, but in a very different fashion than their effect on human macrophages. Thus, lactoferrin peptides limit pro-inflammatory signaling and cytokine production by LPS-activated human macrophages and thereby enhance the resolution of inflammation.

Select targeting of intracellular Toll-interleukin-1 receptor resistance domains for protection against influenza-induced disease

TLRs are a family of PRRs that respond to PAMPs or host-derived Danger-Associated Molecular Patterns (DAMPs) to initiate host inflammation and immune responses. TLR dimerization and recruitment of adapter molecules is critical for intracellular signaling and is mediated through intracellular Toll-Interleukin 1 Receptor Resistance (TIR) domain interactions. Human TIR domains, including reported structures of TIR1, TIR2, TIR6, TIR10, TIRAP, and MyD88, contain Cysteine (Cys) interactions or modifications that are disproportionally at, or near, reported biological TIR interfaces, or in close proximity to functionally important regions. Therefore, we hypothesized that intracellular TIR Cys regulation may have greater functional importance than previously appreciated. Expression of mutant TLR4-C747S or treatment of TLR4 reporter cells with a small molecule, Cys-binding inhibitor of TLR4, TAK-242, abrogated LPS signaling in vitro. Using TAK-242, mice were protected from lethal influenza challenge as previously reported for extracellular TLR4 antagonists. Molecular modeling and sequence analysis of the region surrounding TLR4-Cys747 indicate conservation of a WxxxE motif identified among bacterial and NAD+-consuming TIRs, as well as within the TIRs domains of surface TLRs 1, 2, 4, 6, and 10. Together, these data support the hypothesis that critical Cys within the TIR domain are essential for TLR4 functionality.

Monophosphoryl lipid A induces protection against LPS in medullary thick ascending limb through induction of Tollip and negative regulation of IRAK-1

LPS inhibits [Formula: see text] absorption in the medullary thick ascending limb (MTAL) through a Toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88)-extracellular signal-regulated kinase (ERK) pathway that is upregulated by sepsis. Pretreatment with the nontoxic immunomodulator monophosphoryl lipid A (MPLA) prevents inhibition by LPS through activation of a TLR4-TIR-domain-containing adaptor-inducing interferon-β (TRIF)-phosphatidylinositol 3-kinase (PI3K) pathway that prevents LPS-induced ERK activation. Here, we identified the molecular mechanisms that underlie the protective inhibitory interaction between the MPLA-PI3K and LPS-ERK pathways. Treatment of mouse MTALs with LPS in vitro increased phosphorylation of IL-1 receptor-associated kinase (IRAK)-1, a critical mediator of LPS signaling downstream of TLR4-MyD88. Activation of ERK by LPS was eliminated by a selective IRAK-1 inhibitor, establishing IRAK-1 as the upstream mediator of ERK activation. Pretreatment of MTALs with MPLA in vitro prevented LPS-induced IRAK-1 activation; this effect was dependent on PI3K. Treatment of MTALs with MPLA increased expression of Toll-interacting protein (Tollip), an inducible protein that negatively regulates LPS signaling by inhibiting IRAK-1. The MPLA-induced increase in Tollip protein level was prevented by PI3K inhibitors. In coimmunoprecipitation experiments, MPLA increased the amount of Tollip stably bound to IRAK-1, an interaction that inhibits IRAK-1 activation. These results support a mechanism whereby MPLA increases Tollip expression in the MTAL through a PI3K-dependent pathway. Tollip, in turn, inhibits LPS-induced TLR4 signaling by suppressing activation of IRAK-1, thereby preventing activation of ERK that inhibits [Formula: see text] absorption. These studies show that MPLA induces reprogramming of MTAL cells that protects against LPS stimulation and identify IRAK-1 and Tollip as new therapeutic targets to prevent renal tubule dysfunction in response to infectious and inflammatory stimuli.

Signals trigger state-specific transcriptional programs to support diversity and homeostasis in immune cells

Macrophages play key roles in the immune systems of humans and other mammals. Here, we performed single-cell analyses of the mRNAs and proteins of human macrophages to compare their responses to the signaling molecules lipopolysaccharide (LPS), a component of Gram-negative bacteria, and palmitate (PAL), a free fatty acid. We found that, although both molecules signal through the cell surface protein Toll-like receptor 4 (TLR4), they stimulated the expression of different genes, resulting in specific pro- and anti-inflammatory cellular states for each signal. The effects of the glucocorticoid receptor, which antagonizes LPS signaling, and cyclic AMP–dependent transcription factor 3, which inhibits PAL-induced inflammation, on inflammatory response seemed largely determined by digital on-off events. Furthermore, the quantification of transcriptional variance and signaling entropy enabled the identification of cell state–specific deregulated molecular pathways. These data suggest that the preservation of signaling in distinct cells might confer diversity on macrophage populations essential to maintaining major cellular functions.