A regulatory circuit controlled by extranuclear and nuclear retinoic acid receptor α determines T cell activation and function.

Ligation of retinoic acid receptor alpha (RARα) by RA promotes varied transcriptional programs associated with immune activation and tolerance, but genetic deletion approaches suggest the impact of RARα on TCR signaling. Here, we examined whether RARα would exert roles beyond transcriptional regulation. Specific deletion of the nuclear isoform of RARα revealed an RARα isoform in the cytoplasm of T cells. Extranuclear RARα was rapidly phosphorylated upon TCR stimulation and recruited to the TCR signalosome. RA interfered with extranuclear RARα signaling, causing suboptimal TCR activation while enhancing FOXP3+ regulatory T cell conversion. TCR activation induced the expression of CRABP2, which translocates RA to the nucleus. Deletion of Crabp2 led to increased RA in the cytoplasm and interfered with signalosome-RARα, resulting in impaired anti-pathogen immunity and suppressed autoimmune disease. Our findings underscore the significance of subcellular RA/RARα signaling in T cells and identify extranuclear RARα as a component of the TCR signalosome and a determinant of immune responses.

Algal chloroplast produced camelid VH H antitoxins are capable of neutralizing botulinum neurotoxin.

We have produced three antitoxins consisting of the variable domains of camelid heavy chain-only antibodies (VH H) by expressing the genes in the chloroplast of green algae. These antitoxins accumulate as soluble proteins capable of binding and neutralizing botulinum neurotoxin. Furthermore, they accumulate at up to 5% total soluble protein, sufficient expression to easily produce these antitoxins at scale from algae. The genes for the three different antitoxins were transformed into Chlamydomonas reinhardtii chloroplasts and their products purified from algae lysates and assayed for in vitro biological activity using toxin protection assays. The produced antibody domains bind to botulinum neurotoxin serotype A (BoNT/A) with similar affinities as camelid antibodies produced in Escherichia coli, and they are similarly able to protect primary rat neurons from intoxication by BoNT/A. Furthermore, the camelid antibodies were produced in algae without the use of solubilization tags commonly employed in E. coli. These camelid antibody domains are potent antigen-binding proteins and the heterodimer fusion protein containing two VH H domains was capable of neutralizing BoNT/A at near equimolar concentrations with the toxin. Intact antibody domains were detected in the gastrointestinal (GI) tract of mice treated orally with antitoxin-producing microalgae. These findings support the use of orally delivered antitoxins produced in green algae as a novel treatment for botulism.

Potent intestinal Th17 priming through peripheral lipopolysaccharide-based immunization.

Lipopolysaccharide (LPS) is a potent natural adjuvant, commonly used to amplify Th1 responses. Here, we report that systemic immunization using LPS generates large numbers of specific Th17 cells in murine small intestinal lamina propria. The priming of these Th17 cells required IL-23p19 production by bone marrow-derived cells. In contrast, IL-23 had no impact on Th1 differentiation or overall numbers of Ag-specific regulatory T cells. Experiments using T-cell adoptive transfers revealed a previously unappreciated mechanism for how Th17 responses are amplified in vivo: stimulation through LPS expanded precommitted Th17 cells rather than causing Th17 differentiation. Second, LPS drove Th17 cell expansion independently of IL-23, demonstrating that this cytokine is not necessary for expansion and possibly functions at an earlier stage in Th17 priming. Our data provide an impetus for using LPS-based peripheral vaccination to augment specific T-cell-mediated immunity in the gut mucosa.

The systemic and pulmonary immune response to staphylococcal enterotoxins.

In response to environmental cues the human pathogen Staphylococcus aureus synthesizes and releases proteinaceous enterotoxins. These enterotoxins are natural etiologic entities of severe food poisoning, toxic shock syndrome, and acute diseases. Staphylococcal enterotoxins are currently listed as Category B Bioterrorism Agents by the Center for Disease Control and Prevention. They are associated with respiratory illnesses, and may contribute to exacerbation of pulmonary disease. This likely stems from the ability of Staphylococcal enterotoxins to elicit powerful episodes of T cell stimulation resulting in release of pro-inflammatory cytokines. Here, we discuss the role of the immune system and potential mechanisms of disease initiation and progression.

The oxazolidinone derivative locostatin induces cytokine appeasement.

Damaging inflammation arising from autoimmune pathology and septic responses results in severe cases of disease. In both instances, anti-inflammatory compounds are used to limit the excessive or deregulated cytokine responses. We used a model of robust T cell stimulation to identify new proteins involved in triggering a cytokine storm. A comparative proteomic mining approach revealed the differential mapping of Raf kinase inhibitory protein after T cell recall in vivo. Treatment with locostatin, an Raf kinase inhibitory protein inhibitor, induced T cell anergy by blocking cytokine production after Ag recall. This was associated with a reduction in Erk phosphorylation. Importantly, in vivo treatment with locostatin profoundly inhibited TNF-alpha production upon triggering the Ag-specific T cells. This effect was not limited to a murine model because locostatin efficiently inhibited cytokine secretion by human lymphocytes. Therefore, locostatin should be a useful therapeutic to control inflammation, sepsis, and autoimmune diseases.

The promoter region of the MDR1 gene is largely invariant, but different single nucleotide polymorphism haplotypes affect MDR1 promoter activity differently in different cell lines.

The MDR1 multidrug transporter represents one of the better characterized drug transporters that play an important role in protecting the body against xenobiotic insults. Single nucleotide polymorphisms (SNPs) and SNP haplotypes within this gene have been variously associated with differences in MDR1 expression/function, drug response as well as disease susceptibility. Nonetheless, the effect of polymorphisms at the MDR1 promoter region on its promoter activity remains less characterized. Through the examination of approximately 1.5 kilobases of MDR1 promoter region from five populations, including the Chinese, Malays, Indians, European Americans, and African Americans, we identified eight low-frequency SNPs, of which only two were polymorphic in at least four of the five populations examined. The other SNPs are mainly population-specific, the majority of which occur only in the African-American population. Recapitulation of the various combinations of SNP haplotypes in vitro in promoter-reporter assays revealed a few notable trends. The African and European American-specific haplotypes tended to result in enhanced MDR1 promoter activity only in the human embryonic kidney (HEK) 293 cell line. Haplotype GCTAACC, which occurs at variable frequencies in all the populations examined, with Asians having much lower frequencies (<2%) compared with the European Americans/African Americans (>4%), affected MDR1 promoter activity differently in different cell lines. Compared with the commonest haplotype, GCTA-ACC haplotype resulted in a significant decrease in MDR1 promoter activity in HeLa cells (P < 0.05) but a significant increase in the same promoter activity in HEK293 cells (P < 0.05). These results suggest that the MDR1 promoter region is largely invariant but that different haplotypes have differential effects on the MDR1 promoter activity in different cell lines.

Distinct haplotype profiles and strong linkage disequilibrium at the MDR1 multidrug transporter gene locus in three ethnic Asian populations.

The MDR1 multidrug transporter plays a key role in determining drug bioavailability, and differences in drug response exist amongst different ethnic groups. Numerous studies have identified an association between the MDR1 single nucleotide polymorphism (SNP) exon 26 3435C>T and differences in MDR1 function. We performed a haplotype analysis of the MDR1 gene in three major ethnic groups (Chinese, Malays and Indians) by examining 10 intragenic SNPs. Four were polymorphic in all three ethnic groups: one occurring in the non-coding region and three occurring in coding exons. All three coding SNPs (exon 12 1236C>T, exon 21 2677G>T/A and exon 26 3435C>T) were present in high frequency in each ethnic group, and the derived haplotype profiles exhibited distinct differences between the groups. Fewer haplotypes were observed in the Malays (n = 6) compared to the Chinese (n = 10) and Indians (n = 9). Three major haplotypes (> 10% frequency) were observed in the Malays and Chinese; of these, two were observed in the Indians. Strong linkage disequilibrium (LD) was detected between the three SNPs in all three ethnic groups. The strongest LD was present in the Chinese, followed by Indians and Malays, with the corresponding LD blocks estimated to be approximately 80 kb, 60 kb and 40 kb, respectively. These data strongly support the hypothesis that strong LD between the neutral SNP exon 26 3435C>T and a nearby unobserved causal SNP underlies the observed associations between the neutral SNP and MDR1 functional differences. Furthermore, strong LD between exon 26 3435T and different unobserved causal SNPs in different study populations may provide a plausible explanation for conflicting reports associating the same exon 26 3435T allele with different MDR1 functional changes.