NLRP6 deficiency expands a novel CD103+ B cell population that confers immune tolerance in NOD mice.

Introduction: Gut microbiota have been linked to modulating susceptibility to Type 1 diabetes; however, there are many ways in which the microbiota interact with host cells, including through microbial ligand binding to intracellular inflammasomes (large multi-subunit proteins) to initiate immune responses. NLRP6, a microbe-recognizing inflammasome protein, is highly expressed by intestinal epithelial cells and can alter susceptibility to cancer, obesity and Crohn's disease; however, the role of NLRP6 in modulating susceptibility to autoimmune diabetes, was previously unknown. Methods: We generated NLRP6-deficient Non-obese diabetic (NOD) mice to study the effect of NLRP6-deficiency on the immune cells and susceptibility to Type 1 diabetes development. Results: NLRP6-deficient mice exhibited an expansion of CD103+ B cells and were protected from type 1 diabetes. Moreover, NLRP6-deficient CD103+ B cells express regulatory markers, secreted higher concentrations of IL-10 and TGFb1 cytokines and suppressed diabetogenic T cell proliferation, compared to NLRP6-sufficient CD103+ B cells. Microarray analysis of NLRP6-sufficient and -deficient CD103+ B cells identified 79 significantly different genes including genes regulated by lipopolysaccharide (LPS), tretinoin, IL-10 and TGFb, which was confirmed in vitro following LPS stimulation. Furthermore, microbiota from NLRP6-deficient mice induced CD103+ B cells in colonized NLRP6-sufficient germ-free mice; however, the long-term maintenance of the CD103+ B cells required the absence of NLRP6 in the hosts, or continued exposure to microbiota from NLRP6-deficient mice. Discussion: Together, our data indicate that NLRP6 deficiency promotes expansion and maintenance of a novel TGF -dependent CD103+ Breg population. Thus, targeting NLRP6 therapeutically may prove clinically useful.

Microbial antigen mimics activate diabetogenic CD8 T cells in NOD mice.

Both animal model and human studies indicate that commensal bacteria may modify type 1 diabetes (T1D) development. However, the underlying mechanisms by which gut microbes could trigger or protect from diabetes are not fully understood, especially the interaction of commensal bacteria with pathogenic CD8 T cells. In this study, using islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-reactive CD8 T cell receptor NY8.3 transgenic nonobese diabetic mice, we demonstrated that MyD88 strongly modulates CD8(+) T cell-mediated T1D development via the gut microbiota. Some microbial protein peptides share significant homology with IGRP. Both the microbial peptide mimic of Fusobacteria and the bacteria directly activate IGRP-specific NY8.3 T cells and promote diabetes development. Thus, we provide evidence of molecular mimicry between microbial antigens and an islet autoantigen and a novel mechanism by which the diabetogenicity of CD8(+) T cells can be regulated by innate immunity and the gut microbiota.

The dual effects of B cell depletion on antigen-specific T cells in BDC2.5NOD mice.

B cells play a critical role in the pathogenesis of autoimmune diabetes. To investigate the mechanisms by which B cell depletion therapy attenuates islet ß cell loss and particularly to examine the effect of B cells on both diabetogenic and regulatory Ag-specific T cells, we generated a transgenic BDC2.5NOD mouse expressing human CD20 on B cells. This allowed us to deplete B cells for defined time periods and investigate the effect of B cell depletion on Ag-specific BDC2.5 T cells. We depleted B cells with anti-human CD20 Ab using a multiple injection protocol. We studied two time points, before and after B cell regeneration, to examine the effect on BDC2.5 T cell phenotype and functions that included antigenic response, cytokine profile, diabetogenicity, and suppressive function of regulatory T (T(reg)) cells. We found unexpectedly that B cell depletion induced transient aggressive behavior in BDC2.5 diabetogenic T cells and reduction in T(reg) cell number and function during the depletion period. However, after B cell reconstitution, we found that more regenerated B cells, particularly in the CD1d(-) fraction, expressed immune regulatory function. Our results suggest that the regenerated B cells are likely to be responsible for the therapeutic effect after B cell depletion. Our preclinical study also provides direct evidence that B cells regulate both pathogenic and T(reg) cell function, and this knowledge could explain the increased T cell responses to islet Ag after rituximab therapy in diabetic patients in a recent report and will be useful in design of future clinical protocols.

Identification of a cis-acting element of human dihydrofolate reductase mRNA.

Human dihydrofolate reductase (DHFR) is a critical target in cancer chemotherapy. Previous studies showed that an 82-nt RNA fragment within the DHFR mRNA protein-coding region functions as a DHFR cis-acting response element. In this study, we further investigated the key elements contained within this sequence that are required for the DHFR mRNA-DHFR protein interaction. Using enzymatic foot-printing assays and RNA-binding experiments, we isolated a 27-nt sequence (DHFR27, corresponding to nts 407-433), which bound with high affinity and specificity to human DHFR to form a ribonucleoprotein complex. In vivo transient transfection experiments using a luciferase reporter system revealed that DHFR27 RNA could repress the luciferase expression in a DHFR-dependent manner when placed upstream of luciferase mRNA. This work provides new insights into the essential molecular elements that mediate RNA-protein interactions.

Translational autoregulation of thymidylate synthase and dihydrofolate reductase.

The folate-dependent enzymes, thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are critical for providing the requisite nucleotide precursors for maintaining DNA synthesis and DNA repair. In addition to their essential roles in enzyme catalysis, these two enzymes have now been shown to function as RNA binding proteins. Using in vitro and in vivo experimental model systems, we have shown that the functional consequence of binding of TS protein to its own cognate mRNA, as well as binding of DHFR to its own DHFR mRNA, is translational repression. Herein, we review and update studies focusing on the translational autoregulatory control of TS and DHFR expression and discuss the molecular elements that are required for these specific RNA-protein interactions. Moreover, we present evidence showing that abrogation of these normal translational autoregulatory feedback mechanisms provides the molecular basis for the rapid development of cellular drug resistance.

Characterization of a cis-acting regulatory element in the protein-coding region of human dihydrofolate reductase mRNA.

Previous studies have shown that human DHFR (dihydrofolate reductase), in addition to its critical role in DNA biosynthesis, functions as an RNA-binding protein. The interaction between DHFR and its own mRNA results in translational repression. In this study, we characterized the cis-acting elements on human DHFR mRNA that are required for the DHFR mRNA-DHFR protein interaction. Using a series of gel-shift and nitrocellulose filter-binding assays, a 164 nt RNA sequence, corresponding to nt 401-564, was identified within the coding region that binds to DHFR protein with an affinity similar to that of full-length DHFR mRNA. To document in vivo biological activity, various DHFR sequences contained within the coding region were cloned on to the 5' end of a luciferase reporter plasmid, and transient transfection experiments were performed using human colon cancer RKO cells. In cells transfected with p644/DHFR:401-564, luciferase activity was decreased by 50% when compared with cells transfected with the p644 plasmid alone. Luciferase mRNA levels were identical under each of these conditions, as determined by Northern-blot analysis. In cells transfected with p644/DHFR:401-564, luciferase activity was restored to almost 100% of control when cells were treated with the antifolate analogue methotrexate or with a short-interfering RNA targeting DHFR mRNA. These findings provide evidence that the DHFR 401-564 sequence is a DHFR-response element. In vitro and in vivo studies further localized this cis-element to an 82 nt sequence corresponding to nt 401-482. This work provides new insights into critical elements that mediate RNA-protein interactions.

Identification of critical amino acid residues on human dihydrofolate reductase protein that mediate RNA recognition.

Previous studies have shown that human dihydrofolate reductase (DHFR) acts as an RNA-binding protein, in which it binds to its own mRNA and, in so doing, results in translational repression. In this study, we used RNA gel mobility shift and nitrocellulose filter-binding assays to further investigate the specificity of the interaction between human DHFR protein and human DHFR mRNA. Site-directed mutagenesis was used to identify the critical amino acid residues on DHFR protein required for RNA recognition. Human His-Tag DHFR protein specifically binds to human DHFR mRNA, while unrelated proteins including thymidylate synthase, p53 and glutathione-S-transferase were unable to form a ribonucleoprotein complex with DHFR mRNA. The Cys6 residue is essential for RNA recognition, as mutation at this amino acid with either an alanine (C6A) or serine (C6S) residue almost completely abrogated RNA-binding activity. Neither one of the cysteine mutant proteins was able to repress the in vitro translation of human DHFR mRNA. Mutations at amino acids Ile7, Arg28 and Phe34, significantly reduced RNA-binding activity. An RNA footprinting analysis identified three different RNA sequences, bound to DHFR protein, ranging in size from 16 to 45 nt, while a UV cross-linking analysis isolated an approximately 16 nt RNA sequence bound to DHFR. These studies begin to identify the critical amino acid residues on human DHFR that mediate RNA binding either through forming direct contact points with RNA or through maintaining the protein in an optimal structure that allows for the critical RNA-binding domain to be accessible.

Thymidylate synthase as a translational regulator of cellular gene expression.

Studies from our laboratory have shown that the folate-dependent enzyme, thymidylate synthase (TS), functions as an RNA binding protein. There is evidence that TS, in addition to interacting with its own TS mRNA, forms a ribonucleoprotein complex with a number of other cellular mRNAs, including those corresponding to the p53 tumor suppressor gene and the myc family of transcription factors. Using both in vitro and in vivo model systems, we have demonstrated that the functional consequence of binding of TS protein to its own cognate mRNA, as well as binding of TS to the p53 mRNA, is translational repression. Herein, we review current work on the translational autoregulatory control of TS expression and discuss the molecular elements that are required for the TS protein-TS mRNA interaction. TS may play a critical role in regulating the cell cycle and the process of apoptosis through its regulatory effects on expression of p53 and perhaps other cell cycle related proteins. Finally, the ability of TS to function as a translational regulator may have important consequences with regard to the development of cellular resistance to various anticancer drugs.