SUMMARY Type 1 interferons (IFN) promote inflammation in the skin but the mechanisms responsible for inducing these cytokines are not well understood. We found that IFNβ was abundantly produced by epidermal keratinocytes (KCs) in psoriasis and during wound repair. KC IFNβ production depended on stimulation of mitochondrial antiviral-signaling protein (MAVS) by the antimicrobial peptide LL37 and double stranded-RNA released from necrotic cells. MAVS activated downstream TBK1 (TANK-Binding Kinase 1)-AKT (AKT serine/threonine kinase 1)-IRF3 (interferon regulatory factor 3) signaling cascade leading to IFNβ production, and then promoted maturation of dendritic cells. In mice, the production of epidermal IFNβ by LL37 required MAVS, and human wounded and/or psoriatic skin showed activation of MAVS-associated IRF3 and induction of MAVS and IFNβ gene signatures. These findings show that KCs are an important source of IFNβ and MAVS is critical to this function, and demonstrates how the epidermis triggers unwanted skin inflammation under disease conditions.

Group A Streptococcus (GAS) is among the top 10 causes of infection-related mortality in humans. M protein is the most abundant GAS surface protein, and M1 serotype GAS strains are associated with invasive infections including necrotizing fasciitis and toxic shock syndrome. Here we report that released, soluble M1 protein triggers programmed cell death in macrophages (Mϕ). M1 served as a second signal for caspase-1-dependent NLRP3 inflammasome activation, inducing maturation and release of proinflammatory cytokine IL-1β and macrophage pyroptosis. The structurally dynamic B-repeat domain of M1 was critical for inflammasome activation, which involved K+ efflux and M1 protein internalization by clathrin-mediated endocytosis. Mouse intraperitoneal challenge showed that soluble M1 was sufficient and specific for IL-1β activation, which may represent an early warning to activate host immunity against the pathogen. Conversely, in systemic infection, hyperinflammation associated with M1-mediated pyroptosis and IL-1β release could aggravate tissue injury.

Development of an effective vaccine against the leading human bacterial pathogen group A Streptococcus (GAS) is a public health priority. The species defining group A cell wall carbohydrate (GAC, Lancefield antigen) can be engineered to remove its immunodominant N-acetylglucosamine (GlcNAc) side chain, implicated in provoking autoimmune cross-reactivity in rheumatic heart disease, leaving its polyrhamnose core (GACPR). Here we generate a novel protein conjugate of the GACPR and test the utility of this conjugate antigen in active immunization. Instead of conjugation to a standard carrier protein, we selected SpyAD, a highly conserved GAS surface protein containing both B-cell and T-cell epitopes relevant to the bacterium that itself shows promise as a vaccine antigen. SpyAD was synthesized using the XpressTM cell-free protein expression system, incorporating a non-natural amino acid to which GACPR was conjugated by site-specific click chemistry to yield high molecular mass SpyAD-GACPR conjugates and avoid disruption of important T-cell and B-cell immunological epitopes. The conjugated SpyAD-GACPR elicited antibodies that bound the surface of multiple GAS strains of diverse M types and promoted opsonophagocytic killing by human neutrophils. Active immunization of mice with a multivalent vaccine consisting of SpyAD-GACPR, together with candidate vaccine antigens streptolysin O and C5a peptidase, protected against GAS challenge in a systemic infection model and localized skin infection model, without evidence of cross reactivity to human heart or brain tissue epitopes. This general approach may allow GAC to be safely and effectively included in future GAS subunit vaccine formulations with the goal of broad protection without autoreactivity.

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Selective serotonin reuptake inhibitors (SSRIs) bind 5-HT transporters, leading to the accumulation of 5-HT and amelioration of depression. Although different mouse strains show varying sensitivity to SSRIs in mouse models of depression, the underlying mechanism of these strain differences remains unclear. Here, the SSRI citalopram dose-dependently reduced immobility time in both the FST and TST in DBA/2J mice but not C57BL/6J mice, whereas fluoxetine showed the opposite results. Paroxetine similarly reduced immobility time in both strains. The affinity of citalopram for the 5-HT transporter was 700-fold higher in DBA/2J mice than in C57BL/6J mice, whereas the affinity of fluoxetine was 100-fold higher in C57BL/6J mice than in DBA/2J mice. Furthermore, high citalopram concentrations were required for [3H]5-HT uptake in C57BL/6J but not in DBA/2J mouse cortical synaptosomes, whereas fluoxetine showed the opposite results. The effects of paroxetine on 5-HT transporter binding and synaptosomal 5-HT uptake were similar in the two strains. These results suggest that immobility duration depends on 5-HT transporter binding levels, which lead to apparent strain differences in immobility time in the FST and TST. Furthermore, differences in 5-HT transporter binding may cause variations in SSRI effects on behaviors.