Inflammasomes are multi-protein complexes that function as sensors of endogenous or exogenous damage-associated molecular patterns. Here we show that deficiency of NLRP6 in mouse colonic epithelial cells results in reduced IL-18 levels and altered fecal microbiota characterized by expanded representation of the bacterial phyla Bacteroidetes (Prevotellaceae) and TM7. NLRP6 inflammasome-deficient mice were characterized by spontaneous intestinal hyperplasia, inflammatory cell recruitment, and exacerbation of chemical colitis induced by exposure to dextran sodium sulfate (DSS). Cross-fostering and cohousing experiments revealed that the colitogenic activity of this microbiota is transferable to neonatal or adult wild-type mice, leading to exacerbation of DSS colitis via induction of CCL5. Antibiotic treatment and electron microscopy studies further supported the role of Prevotellaceae as a key representative of this microbiota-associated phenotype. Altogether, perturbations in this inflammasome pathway, including NLRP6, ASC, caspase-1 and IL-18 may constitute a predisposing or initiating event in some cases of human IBD.

Although commensal bacteria are crucial in maintaining immune homeostasis of the intestine, the role of commensal bacteria in immune responses at other mucosal surfaces remains less clear. Here, we show that commensal microbiota composition critically regulates the generation of virus-specific CD4 and CD8 T cells and antibody responses following respiratory influenza virus infection. By using various antibiotic treatments, we found that neomycin-sensitive bacteria are associated with the induction of productive immune responses in the lung. Local or distal injection of Toll-like receptor (TLR) ligands could rescue the immune impairment in the antibiotic-treated mice. Intact microbiota provided signals leading to the expression of mRNA for pro–IL-1β and pro–IL-18 at steady state. Following influenza virus infection, inflammasome activation led to migration of dendritic cells (DCs) from the lung to the draining lymph node and T-cell priming. Our results reveal the importance of commensal microbiota in regulating immunity in the respiratory mucosa through the proper activation of inflammasomes.

SUMMARY Tapasin is a glycoprotein critical for loading Major Histocompatibility Complex (MHC) class I molecules with high affinity peptides. It functions within the multimeric peptide-loading complex (PLC) as a disulfide-linked, stable heterodimer with the thiol oxidoreductase ERp57, and this covalent interaction is required to support optimal PLC activity. Here we present the 2.6 Å resolution structure of the tapasin/ERp57 core of the PLC. The structure reveals the basis for the stable dimerization of tapasin and ERp57 and provides the first example of a protein disulfide isomerase family member interacting with a substrate. Mutational analysis identified a conserved surface on tapasin that interacts with MHC class I molecules and is critical for the peptide loading and editing function of the tapasin-ERp57 heterodimer. By combining the tapasin/ERp57 structure with those of other defined PLC components we present a molecular model that illuminates the processes involved in MHC class I peptide loading.

The presence of a disulfide bond inside the peptide binding groove of MHC class I molecules and of the thiol oxidoreductase ERp57 in the class I loading complex suggests that disulfide bond isomerization may play a role in peptide loading. Here we show that ERp57 and tapasin are disulfide linked inside the loading complex. Mutagenesis of cysteine 95 in tapasin not only abolishes formation of the ERp57-tapasin bond but also prevents complete oxidation of the class I heavy chain in the loading complex. The resulting MHC class I-beta2m heterodimers are poorly loaded with high-affinity peptides in the ER but nevertheless escape to the cell surface where they are unstable. These findings suggest a role for disulfide bond isomerization in tapasin-mediated peptide loading.

In this review, we discuss recent data from our laboratory that address two aspects of major histocompatibility complex (MHC) class I-restricted antigen processing. First, we consider the nature of the peptide-loading complex, which is the assembly of proteins in the endoplasmic reticulum (ER) into which newly synthesized MHC class I-beta(2) microglobulin (beta(2)m) heterodimers are incorporated, and the mechanisms involved in MHC class I assembly and peptide loading that are facilitated by the peptide-loading complex. Second, we discuss mechanisms of cross-presentation, the phenomenon whereby extracellular and luminal protein antigens can be processed by antigen-presenting cells, particularly dendritic cells, and presented by MHC class I molecules to CD8(+) T cells. The focus of the discussion is mainly on the human MHC class I system.

Highlights Saliva has been recommended as an alternative sample for detection of SARS CoV-2 infection, but data are limited. In this study, SARS CoV-2 was detected by RT-PCR in 35 of 124 patients, 30 (85.7 %) by saliva and 33 (94.3 %) by NP swab. The median cycle threshold value was significantly lower for NPS than for saliva. A third of pure saliva samples were difficult to pipet, which slowed processing.

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Peptide binding to MHC class I molecules is a component of a folding and assembly process that occurs in the endoplasmic reticulum (ER) and uses both cellular chaperones and dedicated factors. The involvement of glycoprotein quality-control chaperones and cellular oxidoreductases in peptide binding has led to models that are gradually being refined. Some aspects of the peptide loading process (e.g., the biosynthesis and degradation of MHC class I complexes) conform to models of glycoprotein quality control, but other aspects (e.g., the formation of a stable disulfide-linked dimer between tapasin and ERp57) deviate from models of chaperone and oxidoreductase function. Here we review what is known about the intersection of glycoprotein folding, oxidative reactions, and MHC class I peptide loading, emphasizing events that occur in the ER and within the MHC class I peptide loading complex.