Nucleotide-binding and oligomerization domain-like receptor (NLR) proteins oligomerize into multiprotein complexes termed inflammasomes when activated. Their autoinhibition mechanism remains poorly defined. Here, we report the crystal structure of mouse NLRC4 in a closed form. The adenosine diphosphate-mediated interaction between the central nucleotide-binding domain (NBD) and the winged-helix domain (WHD) was critical for stabilizing the closed conformation of NLRC4. The helical domain HD2 repressively contacted a conserved and functionally important α-helix of the NBD. The C-terminal leucine-rich repeat (LRR) domain is positioned to sterically occlude one side of the NBD domain and consequently sequester NLRC4 in a monomeric state. Disruption of ADP-mediated NBD-WHD or NBD-HD2/NBD-LRR interactions resulted in constitutive activation of NLRC4. Together, our data reveal the NBD-organized cooperative autoinhibition mechanism of NLRC4 and provide insight into its activation.

Plants and animals have evolved structurally related innate immune sensors, designated NLRs, to detect intracellular nonself molecules. NLRs are modular, consisting of N-terminal coiled-coil (CC) or TOLL/interleukin-1 receptor (TIR) domains, a central nucleotide-binding (NB) domain, and C-terminal leucine-rich repeats (LRRs). The polymorphic barley mildew A (MLA) locus encodes CC-containing allelic immune receptors recognizing effectors of the pathogenic powdery mildew fungus. We report the crystal structure of an MLA receptor's invariant CC domain, which reveals a rod-shaped homodimer. MLA receptors also self-associate in vivo, but self-association appears to be independent of effector-triggered receptor activation. MLA CC mutants that fail to self-interact impair in planta cell death activity triggered by the CC domain alone and by an autoactive full-length MLA receptor that mimics its ATP-bound state. Thus, CC domain-dependent dimerization of the immune sensor defines a minimal functional unit and implies a role for the dimeric CC module in downstream immune signaling.

A total of 216 weaning pigs were used in 2 experiments to determine the effects of dietary supplementation of yeast culture (YC) at different dose levels on the growth performance, nutrient digestibility, intestinal morphology, intestinal microflora, and immune response in weanling pigs and to determine whether YC can be a candidate to replace antibiotic growth promoters (AGP). In Exp. 1, 192 pigs (7.5 +/- 0.2 kg of BW) weaned at 28 d of age were randomly allotted to 6 treatments: 1) control (without AGP or YC); 2) AGP (chlortetracycline, 80 mg/kg); 3) 2.5 g/kg of YC (Diamond V XP Yeast Culture); 4) 5 g/kg of YC; 5) 10 g/kg of YC; and 6) 20 g/kg of YC. Each treatment had 8 replicated pens with 4 pigs per pen. Pigs were fed the experimental diets for 21 d. Average daily gain of pigs fed 5 g/kg of YC was greater (P < 0.05) than that of pigs in the control and other YC groups. However, there was no difference between the YC and AGP group. Pigs supplemented with 5 g/kg of YC, 10 g/kg of YC, and AGP had a greater (P < 0.01) ADFI than the control; however, G:F was not affected by treatment. Thus, 5 g/kg of YC supplementation level was chosen for Exp. 2. In Exp. 2, to elucidate the mode of action of YC, 24 nursery pigs (5.8 +/- 0.1 kg of BW; 21 d of age) were randomly allotted into 3 treatments for a 21-d trial. Treatments consisted of 1) control (without AGP or YC), 2) AGP, and 3) 5 g/kg of YC. Blood samples were collected weekly to measure CD4(+), CD8(+) percentage, and blood cytokine content. All pigs were harvested to determine treatment effects on gut microbiota, morphology, and immune function. Dietary supplementation of 5 g/kg of YC improved (P < 0.05) ADG of pigs compared with the control group, but performance of pigs fed YC was similar to those fed AGP. Pigs receiving 5 g/kg of YC had greater (P < 0.05) digestibility of DM, CP, GE, and jejunal villus height and villus height:crypt depth ratio (P < 0.05) compared with pigs fed the control diet. However, no differences in performance, digestibility, or gut morphology were observed between pigs fed YC and AGP. Gut interferon (IFN)-gamma concentrations were greater (P < 0.01) for pigs supplemented with YC compared with control pigs and pigs supplemented with AGP on d 21. However, plasma IFN-gamma concentrations were decreased (P < 0.01) in pigs supplemented with YC and AGP compared with control pigs on d 7, and CD4(+) was decreased (P < 0.01) in pigs supplemented with YC and AGP on d 14. Results indicate that dietary YC supplementation at 5 g/kg had a positive effect on growth performance of nursery pigs by improving jejunal villus height and villus height:crypt depth ratio and by modulating gut immune response. The comparable effect of 5 g/kg of YC supplementation and AGP on the growth performance of nursery pigs indicates that YC may be a good candidate as an antibiotic alternative.

A total of 196 day-old male broiler chicks were randomly allocated to 1 of 4 treatments in a study conducted to determine the effects of dietary supplementation of chito-oligosaccharide (COS) on growth, nutrient digestibility, and serum composition. The experimental diets consisted of an unsupplemented control diet based on corn, soybean meal, and fish meal or similar diets supplemented with either chlortetracycline, 50 mg/kg of COS, or 100 mg/kg of COS. Each treatment was fed to 7 replicate pens of birds, with 7 birds per pen. Broiler performance, nutrient digestibility, cecal microbial concentrations, and serum indices were measured at the end of the starter (d 21) and grower phases (d 42). During the starter period and overall, broilers fed 50 or 100 mg/kg of COS had better (P<0.05) average daily gain, average daily feed intake, and feed conversion than the control birds. The performance of birds fed chlortetracycline was generally intermediate between that of the control and the 2 COS treatments. Compared with the birds in the control or chlortetracycline treatments, the birds receiving 100 mg/kg of COS had better nutrient digestibility of DM, energy, calcium, and phosphorus; higher (P<0.05) concentrations of cecal Lactobacillus; and lower (P<0.05) serum triglyceride and total cholesterol during the starter phase. During the grower phase, the birds fed 100 mg/kg of COS had higher (P<0.05) calcium digestibility and CP retention than those fed the chlortetracycline treatment, and lower concentrations of cecal Escherichia coli than birds in the control treatment. The serum growth hormone level in birds fed 50 mg/kg of COS was higher (P<0.05) than in the other treatments. The birds fed 100 mg/kg of COS had lower (P<0.05) serum triglyceride, higher (P<0.05) serum high-density lipoprotein cholesterol, and higher serum total protein content than birds in the other treatments. In conclusion, dietary supplementation with COS appeared to improve the average daily gain of broilers by increasing the average daily feed intake and nutrient digestibility and modulating the concentrations of cecal microbial flora. Additionally, COS increased serum protein and high-density lipoprotein cholesterol and decreased serum triglyceride.