David R. Peaper

NLRP6 Inflammasome Regulates Colonic Microbial Ecology and Risk for Colitis

Inflammasomes are multiprotein 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 the cytokine, 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.

Microbiota regulates immune defense against respiratory tract influenza A virus infection

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.

Mechanisms of MHC class I-restricted antigen processing and cross-presentation

Summary:  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‐β2 microglobulin (β2m) 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.

Disulfide Bond Isomerization and the Assembly of MHC Class I-Peptide Complexes

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.

Insights into MHC Class I Peptide Loading from the Structure of the Tapasin-ERp57 Thiol Oxidoreductase Heterodimer

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 A resolution structure of the tapasin-ERp57 core of the PLC. The structure revealed that tapasin interacts with both ERp57 catalytic domains, accounting for the stability of the heterodimer, and provided an example of a protein disulfide isomerase family member interacting with substrate. Mutational analysis identified a conserved surface on tapasin that interacted with MHC class I molecules and was critical for peptide loading and editing functions 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.

Regulation of MHC Class I Assembly and Peptide Binding

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.

Tapasin and ERp57 form a stable disulfide-linked dimer within the MHC class I peptide-loading complex

We previously showed that the major histocompatibility complex (MHC) class I chaperone tapasin can be detected as a mixed disulfide with the thiol‐oxidoreductase ERp57. Here we show that tapasin is a unique and preferred substrate, a substantial majority of which is disulfide‐linked to ERp57 within the cell. Tapasin upregulation by interferon‐γ induces sequestration of the vast majority of ERp57 into the MHC class I peptide‐loading complex. The rate of tapasin–ERp57 conjugate formation is unaffected by the absence of β2‐microglubulin (β2m), and is independent of calnexin or calreticulin interactions with monoglucosylated N‐linked glycans. The heterodimer forms spontaneously in vitro upon mixing recombinant ERp57 and tapasin. Noncovalent interactions between the native proteins inhibit the reductase activity of the thioredoxin CXXC motif within the N‐terminal a domain of ERp57 to maintain its interaction with tapasin. Disruption of these interactions by denaturation allows reduction to proceed. Thus, tapasin association specifically inhibits the escape pathway required for disulfide‐bond isomerization within conventional protein substrates, suggesting a specific structural role for ERp57 within the MHC class I peptide‐loading complex.

The redox activity of ERp57 is not essential for its functions in MHC class I peptide loading

ERp57 is an oxidoreductase that, in conjunction with calnexin and calreticulin, assists disulfide bond formation in folding glycoproteins. ERp57 also forms a mixed disulfide with the MHC class I-specific chaperone tapasin, and this dimeric conjugate edits the peptide repertoire bound by MHC class I molecules. In cells unable to form the conjugate, because of tapasin mutation in human studies or ERp57 deletion in mouse studies, peptide loading is impeded. Subtle differences between the mouse and human systems have been observed. Here, we address these differences and expand the analysis to investigate the role of ERp57 redox functions in MHC class I peptide loading. We show in human cells that in the absence of conjugate formation MHC class I recruitment and/or stabilization in the MHC class I peptide-loading complex is impaired, similar to observations in mouse cells. However, we found no role for the enzymatic activities of either the a or a′ domain redox sites of ERp57 in peptide loading. Our data argue that the function of ERp57 in peptide loading is likely caused by other ERp57 functional domains or a combinatorial feature of the tapasin–ERp57 conjugate.

Essential glycan-dependent interactions optimize MHC class I peptide loading

In this study we sought to better understand the role of the glycoprotein quality control machinery in the assembly of MHC class I molecules with high-affinity peptides. The lectin-like chaperone calreticulin (CRT) and the thiol oxidoreductase ERp57 participate in the final step of this process as part of the peptide-loading complex (PLC). We provide evidence for an MHC class I/CRT intermediate before PLC engagement and examine the nature of that chaperone interaction in detail. To investigate the mechanism of peptide loading and roles of individual components, we reconstituted a PLC subcomplex, excluding the Transporter Associated with Antigen Processing, from purified, recombinant proteins. ERp57 disulfide linked to the class I-specific chaperone tapasin and CRT were the minimal PLC components required for MHC class I association and peptide loading. Mutations disrupting the interaction of CRT with ERp57 or the class I glycan completely eliminated PLC activity in vitro. By using the purified system, we also provide direct evidence for a role for UDP-glucose:glycoprotein glucosyltransferase 1 in MHC class I assembly. The recombinant Drosophila enzyme reglucosylated MHC class I molecules associated with suboptimal ligands and allowed PLC reengagement and high-affinity peptide exchange. Collectively, the data indicate that CRT in the PLC enhances weak tapasin/class I interactions in a manner that is glycan-dependent and regulated by UDP-glucose:glycoprotein glucosyltransferase 1.

Rapid Diagnosis of Influenza: State of the Art

Much effort has been expended developing testing modalities for influenza viruses that are capable of providing rapid results to clinicians. Antigen-detection techniques, historically the only methods able to deliver results quickly, are still widely used despite concerns about sensitivity. Recently, nucleic acid amplification tests (NAATs), which can achieve rapid turnaround times and high sensitivity, have become available. In addition, NAATs can detect other respiratory pathogens. Although there are many theoretical advantages to rapid influenza testing, the clinical impact of testing in various patient populations must be considered against the cost and the analytical performance of the tests.