Paradoxical Effects of Endoplasmic Reticulum Aminopeptidase 1 Deficiency on HLA-B27 and Its Role as an Epistatic Modifier in Experimental Spondyloarthritis.

OBJECTIVE: We undertook this study to examine the functional basis for epistasis between endoplasmic reticulum aminopeptidase 1 (ERAP1) and HLA-B27 in experimental spondyloarthritis (SpA). METHODS: ERAP1-knockout rats were created using genome editing and bred with HLA-B27/human ß2 -microglobulin-transgenic (HLA-B27-Tg) rats and HLA-B7-Tg rats. The effects of ERAP1 deficiency on HLA allotypes were determined using immunoprecipitation and immunoblotting, flow cytometry, allogeneic T cell proliferation assays, and gene expression analyses. Animals were examined for clinical features of disease, and tissue was assessed by histology. RESULTS: ERAP1 deficiency increased the ratio of folded to unfolded (ß2 m-free) HLA-B27 heavy chains, while having the opposite effect on HLA-B7. Furthermore, in rats with ERAP1 deficiency, HLA-B27 misfolding was reduced, while free HLA-B27 heavy chain dimers on the cell surface and monomers were increased. The effects of ERAP1 deficiency persisted during up-regulation of HLA-B27 and led to a reduction in endoplasmic reticulum stress. ERAP1 deficiency reduced the prevalence of arthritis in HLA-B27-Tg rats by two-thirds without reducing gastrointestinal inflammation. Dendritic cell abnormalities attributed to the presence of HLA-B27, including reduced allogeneic T cell stimulation and loss of CD103-positive/major histocompatibility complex class II-positive cells, were not rescued by ERAP1 deficiency, while excess Il23a up-regulation was mitigated. CONCLUSION: ERAP1 deficiency reduced HLA-B27 misfolding and improved folding while having opposing effects on HLA-B7. The finding that HLA-B27-Tg rats had partial protection against SpA in this study is consistent with genetic evidence that loss-of-function and/or reduced expression of ERAP1 reduces the risk of ankylosing spondylitis. Functional studies support the concept that the effects of ERAP1 on HLA-B27 and SpA may be a consequence of how peptides affect the biology of this allotype rather than their role as antigenic determinants.

Augmented Renal Clearance Using Population-Based Pharmacokinetic Modeling in Critically Ill Pediatric Patients.

OBJECTIVES: The objectives of this study were to: 1) evaluate the prevalence of augmented renal clearance in critically ill pediatric patients using vancomycin clearance; 2) derive the pharmacokinetic model that best describes vancomycin clearance in critically ill pediatric patients; and 3) correlate vancomycin clearance with creatinine clearance estimated by modified Schwartz or Cockcroft-Gault. DESIGN: Retrospective, two-center, cohort study from 2003 to 2016. SETTING: Clinical drug monitoring services in the PICUs at two tertiary care, teaching hospitals. PATIENTS: Children from 1 to 21 years old. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Identify patients with augmented renal clearance (vancomycin clearance ≥ 130 mL/min/1.73 m used as definition of augmented renal clearance). Derive final population-based pharmacokinetic model and estimate individual patient pharmacokinetic parameters. Compare estimated glomerular filtration rate (modified Schwartz or Cockcroft-Gault depending on age < or ≥ 17 yr) with vancomycin clearance. Augmented renal clearance was identified in 12% of 250 total subjects. The final population-based pharmacokinetic model for vancomycin clearance (L/hr) was 0.118 × weight (e). Median vancomycin clearance in those with versus without augmented renal clearance were 141.3 and 91.7 mL/min/1.73 m, respectively (p < 0.001). By classification and regression tree analysis, patients who were more than 7.9 years old were significantly more likely to experience augmented renal clearance (17% vs 4.6% in those ≤ 7.9 yr old; p = 0.002). In patients with augmented renal clearance, 79% of 29 had vancomycin trough concentrations less than 10 µg/mL, compared with 52% of 221 in those without augmented renal clearance (p < 0.001). Vancomycin clearance was weakly correlated to the glomerular filtration rate estimated by the modified Schwartz or Cockcroft-Gault method (Spearman R = 0.083). CONCLUSIONS: Augmented renal clearance was identified in one of 10 critically ill pediatric patients using vancomycin clearance, with an increase of approximately 50 mL/min/1.73 m in those with augmented renal clearance. As augmented renal clearance results in subtherapeutic antibiotic concentrations, optimal dosing is essential in those exhibiting augmented renal clearance.

The Human Leukocyte Antigen (HLA)-B27 Peptidome in Vivo, in Spondyloarthritis-susceptible HLA-B27 Transgenic Rats and the Effect of Erap1 Deletion.

HLA-B27 is a class I major histocompatibility (MHC-I) allele that confers susceptibility to the rheumatic disease ankylosing spondylitis (AS) by an unknown mechanism. ERAP1 is an aminopeptidase that trims peptides in the endoplasmic reticulum for binding to MHC-I molecules. ERAP1 shows genetic epistasis with HLA-B27 in conferring susceptibility to AS. Male HLA-B27 transgenic rats develop arthritis and serve as an animal model of AS, whereas female B27 transgenic rats remain healthy. We used large scale quantitative mass spectrometry to identify over 15,000 unique HLA-B27 peptide ligands, isolated after immunoaffinity purification of the B27 molecules from the spleens of HLA-B27 transgenic rats. Heterozygous deletion of Erap1, which reduced the Erap1 level to less than half, had no qualitative or quantitative effects on the B27 peptidome. Homozygous deletion of Erap1 affected approximately one-third of the B27 peptidome but left most of the B27 peptidome unchanged, suggesting the possibility that some of the HLA-B27 immunopeptidome is not processed in the presence of Erap1. Deletion of Erap1 was permissive for the AS-like phenotype, increased mean peptide length and increased the frequency of C-terminal hydrophobic residues and of N-terminal Ala, Ser, or Lys. The presence of Erap1 increased the frequency of C-terminal Lys and Arg, of Glu and Asp at intermediate residues, and of N-terminal Gly. Several peptides of potential interest in AS pathogenesis, previously identified in human cell lines, were isolated. However, rats susceptible to arthritis had B27 peptidomes similar to those of non-susceptible rats, and no peptides were found to be uniquely associated with arthritis. Whether specific B27-bound peptides are required for AS pathogenesis remains to be determined. Data are available via ProteomeXchange with identifier PXD005502.

ERAP1 reduces accumulation of aberrant and disulfide-linked forms of HLA-B27 on the cell surface.

OBJECTIVE: Endoplasmic reticulum (ER) aminopeptidase 1 (ERAP1) variants contribute to the risk of ankylosing spondylitis in HLA-B27 positive individuals, implying a disease-related interaction between these gene products. The aim of this study was to determine whether reduced ERAP1 expression would alter the cell surface expression of HLA-B27 and the formation of aberrant disulfide-linked forms that have been implicated in the pathogenesis of spondyloarthritis. METHODS: ERAP1 expression was knocked down in monocytic U937 cells expressing HLA-B27 and endogenous HLA class I. The effect of ERAP1 knockdown on the accumulation HLA-B alleles (B18, B51, and B27) was assessed using immunoprecipitation, isoelectric focusing, and immunoblotting, as well as flow cytometry with antibodies specific for different forms of HLA-B27. Cell surface expression of aberrant disulfide-linked HLA-B27 dimers was assessed by immunoprecipitation and electrophoresis on non-reducing polyacrylamide gels. RESULTS: ERAP1 knockdown increased the accumulation of HLA-B27 on the cell surface including disulfide-linked dimers, but had no effect on levels of HLA-B18 or -B51. Antibodies with unique specificity for HLA-B27 confirmed increased cell surface expression of complexes shown previously to contain long peptides. IFN-γ treatment resulted in striking increases in the expression of disulfide-linked HLA-B27 heavy chains, even in cells with normal ERAP1 expression. CONCLUSIONS: Our results suggest that normal levels of ERAP1 reduce the accumulation of aberrant and disulfide-linked forms of HLA-B27 in monocytes, and thus help to maintain the integrity of cell surface HLA-B27 complexes.

Bayesian Estimation of Vancomycin Pharmacokinetics in Obese Children: Matched Case-Control Study.

PURPOSE: The study objective was to compare different body size descriptors that best estimate vancomycin Vd and clearance (CL). METHODS: Patients between 3 months and 21 years old who received vancomycin for ≥48 hours from 2003 to 2011 were evaluated in this matched case-control study. Cases had body mass index in the ≥85th percentile; controls were nonobese individuals who were matched by age and baseline serum creatinine (SCr). Using a 1-compartment model with first-order kinetics, Bayesian post hoc individual Vd and CL were estimated. FINDINGS: Analysis included 87 matched pairs with 389 vancomycin serum concentrations. Median ages were 10.0 (interquartile range [IQR], 4.8-15.2) years for cases (overweight and obese children) and 10.2 (IQR, 4.5-14.8) years for controls (normal-weight children). Median weights were 44.0 (IQR, 23.4-78.1) kg for cases and 31.3 (IQR, 16.8-47.1) kg for controls. Mean (SD) for the baseline SCr values were also similar between the groups: 0.51 (0.22) (IQR, 0.34-0.67) mg/dL and 0.48 (0.20) (IQR, 0.30-0.60) mg/dL for the cases and controls, respectively. Actual weight and allometric weight (ie, weight(0.75)) were used in the final model to estimate Vd and CL, respectively. The mean Vd and CL, based on weight, for cases were lower than controls by 0.012 L/kg and 0.014 L/kg/h, respectively. IMPLICATIONS: In obese children, actual weight and allometric weight are reasonable, convenient estimations of body fat to use for estimating vancomycin Vd and CL, respectively. However, these pharmacokinetic differences between obese children and those with normal weights are small and may not likely to be clinically relevant in dose variation.

Endoplasmic reticulum aminopeptidase 1 and rheumatic disease: functional variation.

PURPOSE OF REVIEW: To review the recent developments in our understanding of endoplasmic reticulum (ER) aminopeptidase 1 (ERAP1) function in relation to its role in major histocompatibility complex (MHC) class I peptide presentation and human leukocyte antigen (HLA) class I-associated diseases. RECENT FINDINGS: ERAP1 polymorphisms exhibiting loss-of-function have been associated with protection from AS. The aminopeptidase function of ERAP1 optimizes peptides for binding and presentation by MHC class I. Most of the studies have revealed reduced MHC class I expression in situations of reduced ERAP1 function. Under these circumstances, the presented peptides are often N-terminally extended, and cell surface complexes are unstable and fall apart more readily. In contrast, peptides presented by HLA-B*27 : 05 when ERAP1 is silenced are frequently extended on the C-terminus. Recent work has emphasized on the importance of assessing the function of allotypes encoded by ERAP1 haplotypes, rather than effects of single amino acid substitutions. The allotypes found in a series of AS patients were poorer at restoring HLA-B27 expression than allotypes found in unaffected controls, which may seem contrary to the genetic data linking loss-of-function to protection. SUMMARY: More work is needed to understand how ERAP1 variants associated with risk and protection influence the quality and quantity of peptides available for binding to HLA class I molecules in the ER. Moreover, we need to determine allele-specific effects of ERAP1 variants in the context of HLA-B*51 and HLA-Cw*6, which are associated with Behçet's disease and psoriasis, respectively.

HLA-B27 misfolding and ankylosing spondylitis.

Understanding how HLA-B27 contributes to the pathogenesis of spondyloarthritis continues to be an important goal. Current efforts are aimed largely on three areas of investigation; peptide presentation to CD8T cells, abnormal forms of the HLA-B27 heavy chain and their recognition by leukocyte immunoglobulin-like receptors on immune effector cells, and HLA-B27 heavy chain misfolding and intrinsic biological effects on affected cells. In this chapter we review our current understanding of the causes and consequences of HLA-B27 misfolding, which can be defined biochemically as a propensity to oligomerize and form complexes in the endoplasmic reticulum (ER) with the chaperone BiP (HSPA5/GRP78). HLA-B27 misfolding is linked to an unusual combination of polymorphisms that identify this allele, and cause the heavy chain to fold and load peptides inefficiently. Misfolding can result in ER-associated degradation (ERAD) of heavy chains, which is mediated in part by the E3 ubiquitin ligase HRD1 (SYVN1), and the ubiquitin conjugating enzyme UBE2JL. Upregulation of HLA-B27 and accumulation of misfolded heavy chains can activate ER stress signaling pathways that orchestrate the unfolded protein response. In transgenic rats where HLA-B27 is overexpressed, UPR activation is prominent. However, it is specific for heavy chain misfolding, since overexpression of HLA-B7, an allele that does not misfold, fails to generate ER stress. UPR activation has been linked to cytokine dysregulation, promoting lL-23, IFNß, and lL-1α production, and may activate the IL-23/IL-17 axis in these rats. IL-1α and IFNß are pro- and anti-osteoclastogenic cytokines, respectively, that modulate osteoclast development in HLA-B27-expressing transgenic rat monocytes. Translational studies of patient derived cells expressing HLA-B27 at physiologic levels have provided evidence that ER stress and UPR activation can occur in peripheral blood, but this has not been reported to date in isolated macrophages. Inflamed gastrointestinal tissue reveals evidence for HLA-B27 misfolding, ERAD, and autophagy, without acute UPR activation. A more complete picture of conditions that impact HLA-B27 folding and misfolding, the full spectrum and time course of consequences of ER stress, and critical cell types involved is needed to understand the role of HLA-B27 misfolding in spondyloarthritis pathogenesis.

Improved vancomycin dosing in children using area under the curve exposure.

BACKGROUND: : Our objectives were to (1) determine the pharmacokinetic indices of vancomycin in pediatric patients; and (2) compare attainment of 2 target exposures: area under curve (AUC) / minimum inhibitory concentration (MIC) ≥400 and trough concentration ≥15 mcg/mL. METHODS: : The population-based pharmacokinetic modeling was performed using NONMEM 7.2 for children ≥3 months old who received vancomycin for ≥48 hours from 2003 to 2011. A 1-compartment model with first-order kinetics was used to estimate clearance, volume of distribution and AUC. Empiric Bayesian post hoc individual parameters and Monte Carlo simulations (N = 11,000) were performed. RESULTS: : Analysis included 702 patients with 1660 vancomycin serum concentrations. Median age was 6.6 (interquartile range 2.2-13.4) years, weight 22.7 (12.6-46) kg and baseline serum creatinine 0.40 (0.30-0.60) mg/dL. Final model pharmacokinetic indices were clearance (L/h) = 0.248 * Wt * (0.48/serum creatinine) * (ln(age)/7.8) and volume of distribution (L) = 0.636 * Wt. Using these parameters and the observed MIC distribution, Monte Carlo simulation indicated that the initial median dose of 44 (39-52) mg/kg/day was inadequate in most subjects. Regimens of 60 mg/kg/day for subjects ≥12 years old and 70 mg/kg/day for those <12 years old achieved target AUC/MIC in ~75% and trough concentrations ≥15 in ~45% of virtual subjects. An AUC/MIC ~400 corresponded to trough concentration ~8 to 9 mcg/mL. CONCLUSIONS: : Targeted exposure using vancomycin AUC/MIC, compared with trough concentrations, is a more realistic target in children. Depending on age, serum creatinine and MIC distribution, vancomycin in a dosage of 60 to 70 mg/kg/day was necessary to achieve AUC/MIC ≥ 400 in 75% of patients.

Spondylarthritis in HLA-B27/human beta2-microglobulin-transgenic rats is not prevented by lack of CD8.

OBJECTIVE: HLA-B27 predisposes to spondylarthritis by an unknown mechanism. A logical candidate mechanism is through recognition of B27 by CD8+ T cells. The purpose of this study was to examine the effects of a lack of CD8 on the spondylarthritis that develops in B27/human beta(2)-microglobulin (Hubeta(2)m)-transgenic rats. METHODS: A missense mutation in the CD8a gene that causes a loss of CD8alpha expression was identified in offspring of a male Sprague-Dawley rat that had been treated with the mutagen N-ethyl-N-nitrosourea. The mutation was crossed into B27/Hubeta(2)m-transgenic lines on the Lewis background. CD8a(-/-) and CD8a(+/-) progeny were compared on a mixed SD-LEW background as well as after at least 10 backcrosses to LEW rats. CD8 function was assessed by generating cytolytic T lymphocytes (CTLs) against allogeneic DA strain antigens. RESULTS: Homozygous mutant rats showed normal CD8a and CD8b messenger RNA levels but no detectable expression of either protein and an almost complete abrogation of the allogeneic CTL response. Two disease phenotypes previously observed in different B27/Hubeta(2)m-transgenic lines also occurred in the respective CD8a(-/-)-transgenic rat lines. There was no significant difference in disease prevalence or severity between CD8a(-/-) rats and CD8a(+/-) rats. CONCLUSION: All of the previously described disease manifestations in HLA-B27/Hubeta(2)m-transgenic rats arise in the absence of any functional CD8+ T cells. It thus seems unlikely that classic T cell recognition of HLA-B27 is of primary importance in this animal model. The possibility of a secondary role of a CD8-dependent mechanism cannot be entirely excluded.

TNFalpha-induced macrophage death via caspase-dependent and independent pathways.

Macrophages are the principal source of TNFalpha, yet they are highly resistant to TNFalpha-mediated cell death. Previously, employing in vitro differentiated human macrophages, we showed that following the inhibition of NF-kappaB, TNFalpha-induced caspase-8 activation contributes to DNA fragmentation but is not necessary for the loss of the inner mitochondrial transmembrane potential (DeltaPsim) or cell death. We here extend these observations to demonstrate that, when NF-kappaB is inhibited in macrophages, TNFalpha alters lysosomal membrane permeability (LMP). This results in the release of cathepsin B with subsequent loss of DeltaPsim and caspase-8 independent cell death. Interestingly, the cytoprotective, NF-kappaB-dependent protein A20 was rapidly induced in macrophages treated with TNFalpha. Ectopic expression of A20 in macrophages preserves LMP following treatment with TNFalpha, and as a result, mitochondrial integrity is safeguarded and macrophages are protected from cell death. These observations demonstrate that TNFalpha triggers both caspase 8-dependent and -independent cell death pathways in macrophages and identify a novel mechanism by which A20 protects these cells against both pathways.

Additional human beta2-microglobulin curbs HLA-B27 misfolding and promotes arthritis and spondylitis without colitis in male HLA-B27-transgenic rats.

OBJECTIVE: Ankylosing spondylitis and related spondylarthritides are associated with HLA-B27, and also with intestinal inflammation, by unknown mechanisms. The folded HLA-B27 molecule is a trimer of heavy chain, beta2-microglobulin (beta2m), and short peptide. However, B27 heavy chain has an unusual propensity to misfold and trigger the unfolded protein response (UPR). This study was initiated to test the hypothesis that B27 misfolding plays a role in the pathogenesis of spondylarthritis. METHODS: Rats with high transgene copy numbers of HLA-B27 heavy chain together with human beta2m (Hubeta2m) spontaneously develop colitis, peripheral arthritis, and occasional spondylitis, and those with lower transgene copy numbers remain healthy. We crossed disease-prone and healthy HLA-B27/Hubeta2m-transgenic rat lines with a healthy line, 283-2, carrying only the Hubeta2m transgene. HLA-B27 assembly was assessed by pulse-chase analysis of B27 molecules, and UPR triggering was assessed by measuring BiP/Grp78 messenger RNA (mRNA) in splenic concanavalin A blasts. Surface expression of B27 and Hubeta2m was determined by flow cytometry. Disease manifestations were identified by clinical observation, histology, and measurement of cytokine mRNA. RESULTS: The extra Hubeta2m from the 283-2 line significantly reduced B27 misfolding and UPR triggering. Unexpectedly, however, F1 male offspring of the healthy 21-3 line crossed with the 283-2 line showed a high prevalence, severity, and duration of arthritis and spondylitis, in the absence of colitis. The arthropathy showed many features characteristic of human spondylarthritis. CONCLUSION: These results suggest that B27 misfolding is associated with intestinal inflammation, but that neither B27 misfolding nor intestinal inflammation is critical to the development of B27-associated arthropathy.