Tarnjit K. Khera

Fragile X-related protein FXR1 controls post-transcriptional suppression of lipopolysaccharide-induced tumour necrosis factor-alpha production by transforming growth factor-beta1

Tumour necrosis factor‐α (TNF‐α) is a key mediator of inflammation in host defence against infection and in autoimmune disease. Its production is controlled post‐transcriptionally by multiple RNA‐binding proteins that interact with the TNF‐α AU‐rich element and regulate its expression; one of these is Fragile X mental retardation‐related protein 1 (FXR1). The anti‐inflammatory cytokine transforming growth factor‐β1 (TGF‐β1), which is involved in the homeostatic regulation of TNF‐α, causes post‐transcriptional suppression of lipopolysaccharide (LPS)‐induced TNF‐α production. We report here that this depends on FXR1. Using RAW 264.7 cells and bone marrow‐derived macrophages (BMDMϕ) stimulated with LPS and TGF‐β1, we show that TGF‐β1 inhibits TNF‐α protein secretion, whereas TNF‐α mRNA expression remains unchanged. This response is recapitulated by the 3′‐UTR of TNF‐α, which is known to bind FXR1. TGF‐β1 induces FXR1 with a pattern of expression distinct from that of tristetraprolin, T‐cell intracellular antigen 1, or human antigen R. When FXR1 is knocked down, TGF‐β1 is no longer able to inhibit LPS‐induced TNF‐α protein production, and overexpression of FXR1 suppresses LPS‐induced TNF‐α protein production. Targeting the p38 mitogen‐activated protein kinase pathway of LPS‐treated cells with small molecule inhibitors can induce FXR1 protein and mRNA expression. In summary, TGF‐β1 opposes LPS‐induced stabilization of TNF‐α mRNA and reduces the amount of TNF‐α protein, through induction of expression of the mRNA‐binding protein FXR1.

Tissue-Resident Exhausted Effector Memory CD8+ T Cells Accumulate in the Retina during Chronic Experimental Autoimmune Uveoretinitis

Experimental autoimmune uveoretinitis is a model for noninfectious posterior segment intraocular inflammation in humans. Although this disease is CD4+ T cell dependent, in the persistent phase of disease CD8+ T cells accumulate. We show that these are effector memory CD8+ T cells that differ from their splenic counterparts with respect to surface expression of CD69, CD103, and Ly6C. These retinal effector memory CD8+ T cells have limited cytotoxic effector function, are impaired in their ability to proliferate in response to Ag-specific stimulation, and upregulate programmed death 1 receptor. Treatment with fingolimod (FTY720) during the late phase of disease revealed that retinal CD8+ T cells were tissue resident. Despite signs of exhaustion, these cells were functional, as their depletion resulted in an expansion of retinal CD4+ T cells and CD11b+ macrophages. These results demonstrate that, during chronic autoimmune inflammation, exhausted CD8+ T cells become established in the local tissue. They are phenotypically distinct from peripheral CD8+ T cells and provide local signals within the tissue by expression of inhibitory receptors such as programmed death 1 that limit persistent inflammation.

A novel pathogenic RBP-3 peptide reveals epitope spreading in persistent experimental autoimmune uveoretinitis

Experimental autoimmune uveoretinitis (EAU) in the C57BL/6J mouse is a model of non‐infectious posterior segment intraocular inflammation that parallels clinical features of the human disease. The purpose of this study was to analyse the immune response to the four murine subunits of retinol binding protein‐3 (RBP‐3) to identify pathogenic epitopes to investigate the presence of intramolecular epitope spreading during the persistent inflammation phase observed in this model of EAU. Recombinant murine subunits of the RBP‐3 protein were purified and used to immunize C57BL/6J mice to induce EAU. An overlapping peptide library was used to screen RBP‐3 subunit 3 for immunogenicity and pathogenicity. Disease phenotype and characterization of pathogenic subunits and peptides was undertaken by topical endoscopic fundal imaging, immunohistochemistry, proliferation assays and flow cytometry. RBP‐3 subunits 1, 2 and 3 induced EAU in the C57BL/6J mice, with subunit 3 eliciting the most destructive clinical disease. Within subunit 3 we identified a novel uveitogenic epitope, 629–643. The disease induced by this peptide was comparable to that produced by the uveitogenic 1–20 peptide. Following immunization, peptide‐specific responses by CD4+ and CD8+ T‐cell subsets were detected, and cells from both populations were present in the retinal inflammatory infiltrate. Intramolecular epitope spreading between 629–643 and 1–20 was detected in mice with clinical signs of disease. The 629–643 RBP‐3 peptide is a major uveitogenic peptide for the induction of EAU in C57BL/6J mice and the persistent clinical disease induced with one peptide leads to epitope spreading.

Re-programming immunosurveillance in persistent non-infectious ocular inflammation

Ocular function depends on a high level of anatomical integrity. This is threatened by inflammation, which alters the local tissue over short and long time-scales. Uveitis due to autoimmune disease, especially when it involves the retina, leads to persistent changes in how the eye interacts with the immune system. The normal pattern of immune surveillance, which for immune privileged tissues is limited, is re-programmed. Many cell types, that are not usually present in the eye, become detectable. There are changes in the tissue homeostasis and integrity. In both human disease and mouse models, in the most extreme cases, immunopathological findings consistent with development of ectopic lymphoid-like structures and disrupted angiogenesis accompany severely impaired eye function. Understanding how the ocular environment is shaped by persistent inflammation is crucial to developing novel approaches to treatment.

A prospective cohort and extended comprehensive-cohort design provided insights about the generalizability of a pragmatic trial: the ProtecT prostate cancer trial

Objectives Randomized controlled trials (RCTs) deliver robust internally valid evidence but generalizability is often neglected. Design features built into the Prostate testing for cancer and Treatment (ProtecT) RCT of treatments for localized prostate cancer (PCa) provided insights into its generalizability. Study Design and Setting Population-based cluster randomization created a prospective study of prostate-specific antigen (PSA) testing and a comprehensive-cohort study including groups choosing treatment or excluded from the RCT, as well as those randomized. Baseline information assessed selection and response during RCT conduct. Results The prospective study (82,430 PSA-tested men) represented healthy men likely to respond to a screening invitation. The extended comprehensive cohort comprised 1,643 randomized, 997 choosing treatment, and 557 excluded with advanced cancer/comorbidities. Men choosing treatment were very similar to randomized men except for having more professional/managerial occupations. Excluded men were similar to the randomized socio-demographically but different clinically, representing less healthy men with more advanced PCa. Conclusion The design features of the ProtecT RCT provided data to assess the representativeness of the prospective cohort and generalizability of the findings of the RCT. Greater attention to collecting data at the design stage of pragmatic trials would better support later judgments by clinicians/policy-makers about the generalizability of RCT findings in clinical practice.