Photoreceptor Cells Constitutively Express IL-35 and Promote Ocular Immune Privilege

Interleukin-27 is constitutively secreted by microglia in the retina or brain, and upregulation of IL-27 during neuroinflammation suppresses encephalomyelitis and autoimmune uveitis. However, while IL-35 is structurally and functionally similar to IL-27, the intrinsic roles of IL-35 in CNS tissues are unknown. Thus, we generated IL-35/YFP-knock-in reporter mice (p35-KI) and demonstrated that photoreceptor neurons constitutively secrete IL-35, which might protect the retina from persistent low-grade inflammation that can impair photoreceptor functions. Furthermore, the p35-KI mouse, which is hemizygous at the il12a locus, develops more severe uveitis because of reduced IL-35 expression. Interestingly, onset and exacerbation of uveitis in p35-KI mice caused by extravasation of proinflammatory Th1/Th17 lymphocytes into the retina were preceded by a dramatic decrease of IL-35, attributable to massive death of photoreceptor cells. Thus, while inflammation-induced death of photoreceptors and loss of protective effects of IL-35 exacerbated uveitis, our data also suggest that constitutive production of IL-35 in the retina might have housekeeping functions that promote sterilization immunity in the neuroretina and maintain ocular immune privilege

Molecular Characteristics and Efficacy of <i>16D10</i> siRNAs in Inhibiting Root-Knot Nematode Infection in Transgenic Grape Hairy Roots

<div><p>Root-knot nematodes (RKNs) infect many annual and perennial crops and are the most devastating soil-born pests in vineyards. To develop a biotech-based solution for controlling RKNs in grapes, we evaluated the efficacy of plant-derived RNA interference (RNAi) silencing of a conserved RKN effector gene, <i>16D10</i>, for nematode resistance in transgenic grape hairy roots. Two hairpin-based silencing constructs, containing a stem sequence of 42 bp (<i>pART27-42</i>) or 271 bp (<i>pART27-271</i>) of the <i>16D10</i> gene, were transformed into grape hairy roots and compared for their small interfering RNA (siRNA) production and efficacy on suppression of nematode infection. Transgenic hairy root lines carrying either of the two RNAi constructs showed less susceptibility to nematode infection compared with control. Small RNA libraries from four <i>pART27-42</i> and two <i>pART27-271</i> hairy root lines were sequenced using an Illumina sequencing technology. The <i>pART27-42</i> lines produced hundred times more <i>16D10</i>-specific siRNAs than the <i>pART27-271</i> lines. On average the <i>16D10</i> siRNA population had higher GC content than the <i>16D10</i> stem sequences in the RNAi constructs, supporting previous observation that plant dicer-like enzymes prefer GC-rich sequences as substrates for siRNA production. The stems of the <i>16D10</i> RNAi constructs were not equally processed into siRNAs. Several hot spots for siRNA production were found in similar positions of the hairpin stems in <i>pART27-42</i> and <i>pART27-271</i>. Interestingly, stem sequences at the loop terminus produced more siRNAs than those at the stem base. Furthermore, the relative abundance of guide and passenger single-stranded RNAs from putative siRNA duplexes was largely correlated with their 5′ end thermodynamic strength. This study demonstrated the feasibility of using a plant-derived RNAi approach for generation of novel nematode resistance in grapes and revealed several interesting molecular characteristics of transgene siRNAs important for optimizing plant RNAi constructs.</p></div

Putative <i>pART27-42</i>-derived siRNA duplexes, their 5′ end strength, and the reads of passenger and guide ssRNAs.

1<p>The 5′ end strength (four or two base parings and one 3′ overhang) was calculated following the methods of Freier et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069463#pone.0069463-Freier1" target="_blank">[44]</a> and Hutvagner <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069463#pone.0069463-Hutvagner1" target="_blank">[43]</a>.</p>2<p>The nucleotides in italic were derived from the loop sequence of <i>pART27-42</i>.</p>3<p>The siRNA duplexes, which followed the 5′ end strength rule (see text for detail) for producing guide and passenger ssRNAs, were in bold (4 bp calculation) or underlined (2 bp calculation).</p>4<p>“Rn” refers to small RNAs which were not among the top 50 small RNAs listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069463#pone-0069463-t002" target="_blank">Table 2</a>.</p

Reproduction of root-knot nematodes on <i>16D10</i> transgenic hairy root lines.

<p>(A) Eggs per hairy root. (B) Eggs per gram hairy root. NC is the negative control (<i>pART27</i> 0). 42-L1 and 271-L5 represent the abbreviations of <i>pART27-42</i> line 1 and <i>pART27-271</i> line 5, respectively. Bars represent the means±SEs observed from individual hairy root lines. Bars (hairy root lines) with “Δ” were significantly different from the negative control at P<0.01, on the basis of log₁₀ transformed data. Data for this figure were provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069463#pone.0069463.s001" target="_blank">Table S1</a>.</p

Tracking the role of Aire in immune tolerance to the eye with a TCR transgenic mouse model.

Roughly one-half of mice with partial defects in two immune tolerance pathways (AireGW/+Lyn-/- mice) spontaneously develop severe damage to their retinas due to T cell reactivity to Aire-regulated interphotoreceptor retinoid-binding protein (IRBP). Single-cell T cell receptor (TCR) sequencing of CD4+ T cells specific for a predominate epitope of IRBP showed a remarkable diversity of autoantigen-specific TCRs with greater clonal expansions in mice with disease. TCR transgenic mice made with an expanded IRBP-specific TCR (P2.U2) of intermediate affinity exhibited strong but incomplete negative selection of thymocytes. This negative selection was absent in IRBP-/- mice and greatly defective in AireGW/+ mice. Most P2.U2+/- mice and all P2.U.2+/-AireGW/+ mice rapidly developed inflammation of the retina and adjacent uvea (uveitis). Aire-dependent IRBP expression in the thymus also promoted Treg differentiation, but the niche for this fate determination was small, suggesting differences in antigen presentation leading to negative selection vs. thymic Treg differentiation and a stronger role for negative selection in preventing autoimmune disease in the retina

Sequences, GC content and reads for the top 50 small RNAs from four <i>pART27-42</i> hairy root lines.

1<p>Nucleotides in italic were derived from the loop sequence of <i>pART27-42</i>.</p

Distribution of the <i>16D10</i> small RNAs along the <i>pART27-271</i> hairpin stem.

<p>The schematic hairpin structure of the <i>pART27-271</i> construct is presented with the green line representing the sense strand, the red line representing the antisense strand, and the blue open circle representing the 39 nt loop. The numbers “100” and “200” along the stem indicate nucleotide positions from the 5′ stem end. The <i>16D10</i> 42 bp core coding region is marked as a purple box on the stem. GC content was marked for the stem base region, the middle core region, and loop terminus region. Each small RNA is represented as a block/line, with the thickness of a block/line indicating the relative abundance of a particular small RNA. Passenger and guide ssRNAs were aligned along the sense and antisense strands, respectively. Data for this figure were provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069463#pone.0069463.s004" target="_blank">Table S4</a>.</p

Numbers of <i>16D10</i> small RNA reads observed from four <i>pART27-42</i> and two <i>pART27-271</i> transgenic hairy root lines.

<p>Numbers of <i>16D10</i> small RNA reads observed from four <i>pART27-42</i> and two <i>pART27-271</i> transgenic hairy root lines.</p

Representative transgenic grape hairy root lines used in this study.

<p>Individual hairy root lines carrying a <i>16D10</i> RNAi construct, <i>pART27-42</i> or <i>pART27-271</i>, were cultured and inoculated with J2 RKNs to evaluate their resistance against RKNs. <i>pART27 0</i> was a control line which was transformed with an empty binary vector <i>pART27</i>. Note that <i>pART27-271</i> line 20 and <i>pART27-42</i> line 24 showed contrasting variation in their root morphology and proliferation. The pictures were taken three weeks (<i>pART27-271</i> line 20 and <i>pART27-42</i> line 24) or five weeks (the rest) after nematode inoculation.</p