Colonizing enteric bacteria are shown to inhibit the antimicrobial process of host cell apoptosis through the action of NleB1, a type III secretion system effector with N-acetylglucosamine transferase activity, which can bind and modify eukaryotic death-domain-containing proteins. Previous studies identified a group of effectors from enteropathogenic Escherichia coli that can inhibit host nuclear factor-κB (NF-κB) signalling, yet only one of them, known as NleB, is required for bacterial virulence in vivo. Two papers published in this issue demonstrate the unique mechanism of action of NleB. It directly targets death receptor signalling complexes, binding to the death domains (DDs) of multiple DD-containing proteins including the TNF receptor, FAS, RIPK1, TRADD and FADD. NleB is shown to function as an N-acetylglucosamine (GlcNAc) transferase that modifies a conserved DD arginine, blocking the receptor-adapter interaction. These findings suggest that GlcNAc modification is essential for bacterial virulence and can regulate death receptor signalling. Successful infection by enteric bacterial pathogens depends on the ability of the bacteria to colonize the gut, replicate in host tissues and disseminate to other hosts. Pathogens such as Salmonella, Shigella and enteropathogenic and enterohaemorrhagic (EPEC and EHEC, respectively) Escherichia coli use a type III secretion system (T3SS) to deliver virulence effector proteins into host cells during infection that promote colonization and interfere with antimicrobial host responses1,2,3. Here we report that the T3SS effector NleB1 from EPEC binds to host cell death-domain-containing proteins and thereby inhibits death receptor signalling. Protein interaction studies identified FADD, TRADD and RIPK1 as binding partners of NleB1. NleB1 expressed ectopically or injected by the bacterial T3SS prevented Fas ligand or TNF-induced formation of the canonical death-inducing signalling complex (DISC) and proteolytic activation of caspase-8, an essential step in death-receptor-induced apoptosis. This inhibition depended on the N-acetylglucosamine transferase activity of NleB1, which specifically modified Arg 117 in the death domain of FADD. The importance of the death receptor apoptotic pathway to host defence was demonstrated using mice deficient in the FAS signalling pathway, which showed delayed clearance of the EPEC-like mouse pathogen Citrobacter rodentium and reversion to virulence of an nleB mutant. The activity of NleB suggests that EPEC and other attaching and effacing pathogens antagonize death-receptor-induced apoptosis of infected cells, thereby blocking a major antimicrobial host response.

Renal inflammation, leading to fibrosis and impaired function is a major contributor to the development of hypertension. The NLRP3 inflammasome mediates inflammation in several chronic diseases by processing the cytokines pro-interleukin (IL)-1β and pro-IL-18. In this study, we investigated whether MCC950, a recently-identified inhibitor of NLRP3 activity, reduces blood pressure (BP), renal inflammation, fibrosis and dysfunction in mice with established hypertension. C57BL6/J mice were made hypertensive by uninephrectomy and treatment with deoxycorticosterone acetate (2.4 mg/d, s.c.) and 0.9% NaCl in the drinking water (1K/DOCA/salt). Normotensive controls were uninephrectomised and received normal drinking water. 10 days later, mice were treated with MCC950 (10 mg/kg/d, s.c.) or vehicle (saline, s.c.) for up to 25 days. BP was monitored by tail-cuff or radiotelemetry; renal function by biochemical analysis of 24-hour urine collections; and kidney inflammation/pathology was assessed by real-time PCR for inflammatory gene expression, flow cytometry for leukocyte influx and Picrosirius red histology for collagen. Over the 10 days post-surgery, 1K/DOCA/salt-treated mice became hypertensive, developed impaired renal function, and displayed elevated renal levels of inflammatory markers, collagen and immune cells. MCC950 treatment from day 10 attenuated 1K/DOCA/salt-induced increases in renal expression of inflammasome subunits (NLRP3, ASC, pro-caspase-1) and inflammatory/injury markers (pro-IL-18, pro-IL-1β, IL-17A, TNF-α, osteopontin, ICAM-1, VCAM-1, CCL2, vimentin), each by 25-40%. MCC950 reduced interstitial collagen and accumulation of certain leukocyte subsets in kidneys of 1K/DOCA/salt-treated mice, including CD206⁺ (M2-like) macrophages and interferon-gamma-producing T cells. Finally, MCC950 partially reversed 1K/DOCA/salt-induced elevations in BP, urine output, osmolality, [Na⁺] and albuminuria (each by 20-25%). None of the above parameters were altered by MCC950 in normotensive mice. MCC950 was effective at reducing BP and limiting renal inflammation, fibrosis and dysfunction in mice with established hypertension. This study provides proof-of-concept that pharmacological inhibition of the NLRP3 inflammasome is a viable anti-hypertensive strategy.

Innate immune responses triggered by the prototypical inflammatory stimulus LPS are mediated by TLR4 and involve the coordinated production of a multitude of inflammatory mediators, especially IL-6, which signals via the shared IL-6 cytokine family receptor subunit gp130. However, the exact role of IL-6, which can elicit either proinflammatory or anti-inflammatory responses, in the pathogenesis of TLR4-driven inflammatory disorders, as well as the identity of signaling pathways activated by IL-6 in a proinflammatory state, remain unclear. To define the contribution of gp130 signaling events to TLR4-driven inflammatory responses, we combined genetic and therapeutic approaches based on a series of gp130^F/F knock-in mutant mice displaying hyperactivated IL-6–dependent JAK/STAT signaling in an experimental model of LPS/TLR4-mediated septic shock. The gp130^F/F mice were markedly hypersensitive to LPS, which was associated with the specific upregulated production of IL-6, but not TNF-α. In gp130^F/F mice, either genetic ablation of IL-6, Ab-mediated inhibition of IL-6R signaling or therapeutic blockade of IL-6 trans-signaling completely protected mice from LPS hypersensitivity. Furthermore, genetic reduction of STAT3 activity in gp130^F/F:Stat3^+/− mice alleviated LPS hypersensitivity and reduced LPS-induced IL-6 production. Additional genetic approaches demonstrated that the TLR4/Mal pathway contributed to LPS hypersensitivity and increased IL-6 production in gp130^F/F mice. Collectively, these data demonstrate for the first time, to our knowledge, that IL-6 trans-signaling via STAT3 is a critical modulator of LPS-driven proinflammatory responses through cross-talk regulation of the TLR4/Mal signaling pathway, and potentially implicate cross-talk between JAK/STAT and TLR pathways as a broader mechanism that regulates the severity of the host inflammatory response.

Activin A is a member of the transforming growth factor-β superfamily, which we have identified as having a role in inflammatory responses. We show that circulating levels of activin increase rapidly after LPS-induced challenge through activation of Toll-like receptor 4 and the key adaptor protein, MyD88. Treatment with the activin-binding protein, follistatin, alters the profiles of TNF, IL-1β, and IL-6 after LPS stimulation, indicating that activin modulates the release of several key proinflammatory cytokines. Further, mice administered one 10-μg dose of follistatin to block activin effects have increased survival after a lethal dose of LPS, and the circulating levels of activin correlate with survival outcome. These findings demonstrate activin A's crucial role in the inflammatory response and show that blocking its actions by the use of follistatin has significant therapeutic potential to reduce the severity of inflammatory diseases.