Aqueous Tear Deficiency Increases Conjunctival Interferon-c (IFN-c) Expression and Goblet Cell Loss

PURPOSE. To investigate the hypothesis that increased interferon-c (IFN-c) expression is associated with conjunctival goblet cell loss in subjects with tear dysfunction. METHODS. Goblet cell density (GCD) was measured in impression cytology from the temporal bulbar conjunctiva, and gene expression was measured in cytology samples from the nasal bulbar conjunctiva obtained from 68 subjects, including normal control, meibomian gland disease (MGD), non-Sjögren syndrome (non-SSATD)-, and Sjögren syndrome (SSATD)-associated aqueous tear deficiency. Gene expression was evaluated by real-time PCR. Tear meniscus height (TMH) was measured by optical coherence tomography. Fluorescein and lissamine green dye staining evaluated corneal and conjunctival disease, respectively. Between-group mean differences and correlation coefficients were calculated. RESULTS. Compared to control, IFN-c expression was significantly higher in both ATD groups, and its receptor was higher in SSATD. Expression of IL-13 and its receptor was similar in all groups. Goblet cell density was lower in the SSATD group; expression of MUC5AC mucin was lower and cornified envelope precursor small proline-rich region (SPRR)-2G higher in both ATD groups. Interferon-c transcript number was inversely correlated with GCD (r ¼ À0.37, P < 0.04) and TMH (r ¼ À0.37, P ¼ 0.02), and directly correlated with lissamine green staining (r ¼ 0.51, P < 0.001) and SPRR-2G expression (r ¼ 0.32, P < 0.05). CONCLUSIONS. Interferon-c expression in the conjunctiva was higher in aqueous deficiency and correlated with goblet cell loss and severity of conjunctival disease. These results support findings of animal and culture studies showing that IFN-c reduces conjunctival goblet cell number and mucin production

The Nicotinic Acetylcholine Receptor Dα7 Is Required for an Escape Behavior inDrosophila

Acetylcholine is the major excitatory neurotransmitter in the central nervous system of insects. Mutant analysis of the Dα7 nicotinic acetylcholine receptor (nAChR) ofDrosophila shows that it is required for the giant fiber-mediated escape behavior. The Dα7 protein is enriched in the dendrites of the giant fiber, and electrophysiological analysis of the giant fiber circuit showed that sensory input to the giant fiber is disrupted, as is transmission at an identified cholinergic synapse between the peripherally synapsing interneuron and the dorsal lateral muscle motor neuron. Moreover, we found thatgfA(1), a mutation identified in a screen for giant fiber defects more than twenty years ago, is an allele ofDα7. Therefore, a combination of behavioral, electrophysiological, anatomical, and genetic data indicate an essential role for the Dα7 nAChR in giant fiber-mediated escape inDrosophila

Genomic Structure and Mutational Analysis of the<i>Dα7</i> Gene

<div><p>(A) The<i>Dα7</i> gene consists of 16 exons. The 5′ and 3′UTRs are drawn in blue, and the ORF is colored red. The insertion sites of the three P-elements are shown above the gene, while the extent of each deletion generated by imprecise excision of the P-elements is depicted below.</p> <p>(B) The structural domains of the Dα7 protein are shown in a schematic representation, and the minimum extent of the lesion associated with the two deletions that remove part of the ORF are indicated by brackets, (), below. Abbreviations: LBD, ligand-binding domain; M1–M4, transmembrane domains 1–4; SP, signal peptide.</p> <p>(C) Immunostaining using an anti-Dα7 antibody is absent in<i>PΔEY6</i>. Here representative staining in the medulla is shown. The precise excision<i>PΔEY5</i> (left image) was used as a control, and whole-mount of the mutant and control brains were processed together. Scale bar, 20 μm.</p></div

gfA¹ is a Mutant Allele of<i>Dα7</i>

<div><p>(A) The response of transheterozygous animals to giant fiber stimulation at 1, 10, and 100 Hz is shown as a histogram using Canton-S (<i>CS</i>) flies as controls.<i>p</i>-Values (1 Hz):<i>CS</i> (<i>n</i> = 6);<i>gfA¹,</i> 0.004 (<i>n</i> = 6);<i>gfA¹/PΔD5,</i> 0.0009 (<i>n</i> = 4);<i>gfA¹/PΔEY6,</i> 0.02 (<i>n</i> = 5).<i>p</i>-Values for 10 Hz and 100 Hz were 2.8 × 10⁻⁵ for all mutant genotypes.</p> <p>(B)<i>gfA¹</i> flies do not show loss of Dα7 protein. Representative staining in the medulla of<i>CS</i> and<i>gfA¹</i> flies is shown. Control and mutant flies were processed together for immunohistochemistry. Scale bar: 20 μm.</p> <p>(C)<i>gfA¹</i> and<i>PΔEY6</i> show dominant phenotype at the PSI-DLM synapse. Comparison of the response to 100 Hz giant fiber stimulation of<i>PΔD5, PΔEY6,</i> and<i>gfA¹</i> in transheterozygous combinations with<i>CS</i> flies as controls is shown as a histogram.<i>p</i>-Values:<i>CS</i> (<i>n</i> = 6),<i>PΔD5</i>/+<i>,</i> 0.99 (<i>n</i> = 6);<i>PΔEY6</i>/+<i>,</i> 0.0002 (<i>n</i> = 7);<i>gfA¹</i>/+<i>,</i> 0.0001 (<i>n</i> = 5).</p> <p>(D)<i>gfA¹</i> flies are dominant in jump behavior. Comparison of jump behavior in<i>bw; st, PΔEY6/+; bw; st,</i> and<i>gfA¹/+; bw; st</i> is shown in a histogram.<i>gfA¹/+; bw; st</i> flies fail to jump while<i>bw; st</i> and<i>PΔEY6/+; bw; st</i> show no significant difference.<i>p</i>-Values:<i>bw; st</i> (<i>n</i> = 10),<i>PΔEY6/+; bw; st</i> (<i>n</i> = 10), 0.23,<i>gfA¹ /+; bw; st</i>, (<i>n</i> = 10), 0.00001.</p> <p>(E) Electropherogram showing basepair change that results in amino acid substitution in<i>gfA¹</i> mutant relative to the<i>CS</i> (wt) sequence.</p> <p>(F) Location of mutated residue in<i>gfA¹</i> (shown in red) indicated on the structure of the nAChR fromTorpedo marmorata [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040063#pbio-0040063-b035" target="_blank">35</a>]. This protein consists of five subunits, one of which is depicted in blue, while the others are in cyan. The residue is drawn in van der Waals representation to emphasize its position. This figure was generated using VMD [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040063#pbio-0040063-b056" target="_blank">56</a>].</p></div

Intracellular Recordings from DLMmn

<div><p>(A) mCD8-GFP driven by<i>Dα7</i> GAL4 labels the DLMmns including the dorsal MN5 (arrows). Scale bar: 50 μm.</p> <p>(B) Experimental method for recording from MN5. The fly is injected in DLMs with Texas red dextran, which is then allowed to be transported back to the cell body (left). Scale bar: 50 μm. Whole cell patch clamp recordings are made from the cell body after dissection to remove the asynchronous flight muscles and the gut. The giant fiber was stimulated through insulated tungsten electrodes placed in the eyes.</p> <p>(C) The backfilled MN5 is shown on the left. The organization of the branch of the giant fiber circuit supplying DLMs is depicted on the right. Synapses between the giant fiber and PSI and between the PSI and DLMmn are circled and indicated with arrowheads. Abbreviations: ADMN, anterior dorsal motor nerve; GF, giant fiber; PDMN, posterior dorsal motor nerve.</p> <p>(D) Response of DLMmn to current injection is shown. Current pulses in steps of 200 pA were applied starting at −500 pA.</p> <p>(E) The top tracings show recordings from MN5 in response to 1-Hz giant fiber stimulation. The middle tracings show stimulation at 10 Hz. The bottom tracings show several overlaid EPSPs at higher magnification.</p></div

Defective Neurotransmission at the PSI-DLMmn Synapse in<i>Dα7</i> Mutants

<div><p>The response of the DLMs to direct activation of the giant fiber is shown. The allele<i>PΔL1</i> served as control for<i>PΔ14G, PΔ41,</i> and<i>PΔD5,</i> while<i>PΔEY5</i> served as control for<i>PΔEY6</i>. For rescue of the<i>PΔD5</i> and<i>PΔEY6</i> mutant alleles, experiments were performed in males with genotype<i>PΔD5/Y; UAS Dα7/OK307 GAL4</i> and<i>PΔEY6/Y; UAS Dα7/OK307 GAL4,</i> respectively.</p> <p>(A) Schematic representation of the giant fiber circuit. Visual and mechanosensory input is transmitted via mixed electrical and chemical synapses to the giant fiber, which carries it to the thoracic ganglion. The TTMmn and PSI neurons are connected via electrical synapses to the giant fiber, and the PSI makes a chemical synapse onto the axon of the DLMmns (encircled).</p> <p>(B) Representative traces of intracellular recordings from DLM muscles for<i>PΔEY5, PΔEY6,</i> and<i>PΔEY6/Y; UAS Dα7/OK307 GAL4</i>.</p> <p>(C) The response of the mutant and control alleles to giant fiber stimulation is summarized in histograms. The top bar graph shows the average latency of the responses at 1 Hz. The second, third, and fourth bar graphs show the number of responses to ten stimuli at 1 Hz, 10 Hz, and 100 Hz respectively. The<i>p</i>-values are as follows. For 1 Hz:<i>PΔL1, n</i> = 6;<i>PΔ14G,</i> 1 (<i>n</i> = 6);<i>PΔ41,</i> 0.04 (<i>n</i> = 7);<i>PΔD5,</i> 2 × 10⁻⁶ (<i>n</i> = 5);<i>PΔD5/Y; UAS Dα7/OK307 GAL4,</i> 1 (<i>n</i> = 4);<i>PΔEY5, n</i> = 5;<i>PΔEY6,</i> 2 × 10⁻⁶ (<i>n</i> = 9);<i>PΔEY6/Y</i>;<i>UAS Dα7/OK307 GAL4,</i> 1 (<i>n</i> = 5). For 10 Hz:<i>PΔL1, n</i> = 6;<i>PΔ14G,</i> 1 (<i>n</i> = 6);<i>PΔ41,</i> 2 × 10⁻⁶ (<i>n</i> = 7);<i>PΔD5,</i> 2 × 10⁻⁶ (<i>n</i> = 5);<i>PΔD5/Y; UAS Dα7/OK307 GAL4,</i> 1 (<i>n</i> = 4);<i>PΔEY5, n</i> = 5;<i>PΔEY6,</i> 2 × 10⁻⁶ (<i>n</i> = 9);<i>PΔEY6/Y; UAS Dα7/OK307 GAL4,</i> 1 (<i>n</i> = 5). For 100 Hz:<i>PΔL1, n</i> = 6;<i>PΔ14G,</i> 4 × 10⁻⁶ (<i>n</i> = 6);<i>PΔ41,</i> 2 × 10⁻⁶ (<i>n</i> = 7);<i>PΔD5,</i> 2 × 10⁻⁶ (<i>n</i> = 5);<i>PΔD5/Y; UAS Dα7/OK307 GAL4,</i> 0.9 (<i>n</i> = 4);<i>PΔEY5, n</i> = 5;<i>PΔEY6,</i> 2 × 10⁻⁶ (<i>n</i> = 9);<i>PΔEY6/Y; UAS Dα7/OK307 GAL4,</i> 0.7 (<i>n</i> = 5). All error bars represent SEM. The<i>p</i>-values for latency measurements at 1 Hz are:<i>PΔL1,</i> (<i>n</i> = 5);<i>PΔ14G,</i> 1 (<i>n</i> = 6);<i>PΔ41,</i> 8.6 × 10⁻⁵ (<i>n</i> = 5);<i>PΔD5,</i> 3.2 × 10⁻⁵ (<i>n</i> = 2);<i>PΔD5/Y</i>;<i>UAS Dα7/OK307 GAL4,</i> 1 (<i>n</i> = 4);<i>PΔEY5,</i> (<i>n</i> = 5);<i>PΔEY6,</i> 0.02 (<i>n</i> = 3);<i>PΔEY6/Y</i>;<i>UAS Dα7/OK307 GAL4,</i> 1 (<i>n</i> = 5).</p> <p>(D) The top panel shows representative intracellular recording from the DLM with direct stimulation of its motor neuron at 1, 10, and 100 Hz. The bottom traces show representative recording from TTM with activation of the giant fiber.</p></div

Behavioral Characterization of<i>Dα7</i> Mutations

<div><p>The response of flies in a variety of behavioral assays is shown in this figure. The error bars represent SEM. In all assays, the precise excision of P-element line KG3295,<i>PΔL1,</i> served as a control for the mutant lines<i>PΔ14G, PΔ41,</i> and<i>PΔD5</i>. The precise excision of line EY10801,<i>PΔEY5,</i> served as a control for<i>PΔEY6</i>.</p> <p>(A) Flight test. Flies were individually dropped in a plastic cylinder, and the height at which they landed was recorded. Ten flies were tested for each genotype.</p> <p>(B) Olfactory response. Ten flies were placed in a Petri dish containing traps baited with food, and the number of trapped flies was recorded. Averages were calculated from between eight and ten trials for each genotype.</p> <p>(C) Visually mediated jump assay. Flies were placed in a Petri dish illuminated with green LEDs. A lights-off stimulus was presented by turning off the LEDs for 20 ms. For this assay it was necessary to use white-eyed flies. Since<i>PΔEY5</i> and<i>PΔEY6</i> are red-eyed, we placed them in a<i>bw; st</i> background, which produces white-eyed flies.<i>p</i>-Values:<i>PΔ14G,</i> 1.9 × 10⁻⁶ (<i>n</i> = 9);<i>PΔ41,</i> 1.9 × 10⁻⁶ (<i>n</i> = 11);<i>PΔD5,</i> 1.9 × 10⁻⁶ (<i>n</i> = 9);<i>PΔEY6,</i> 1.9 × 10⁻⁶ (<i>n</i> = 7).</p> <p>(D) Representative ERGs are shown for control and mutant alleles. We recorded extracellular responses from the eye to 1-s pulses of white light. The timing of the light pulse is shown below each voltage trace.</p> <p>(E) Visual performance was tested using the counter-current assay of Benzer [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040063#pbio-0040063-b029" target="_blank">29</a>].<i>p</i>-Values:<i>PΔ14G,</i> 0.22 (<i>n</i> = 5);<i>PΔ41,</i> 0.02 (<i>n</i> = 5);<i>PΔD5,</i> 0.003 (<i>n</i> = 5);<i>PΔEY6,</i> 1 (<i>n</i> = 5).</p></div

Long-Latency Response of TTM to Giant Fiber Stimulation

<div><p>(A) Recording configuration. The giant fiber was stimulated via tungsten electrodes in the eyes, and extracellular potentials were recorded from the TTM.</p> <p>(B) Average long- and short-latency response of TTM to giant fiber stimulation in different genotypes.<i>PΔD5 rescue</i> and<i>PΔEY6 rescue</i> had genotypes<i>PΔD5/Y; UAS Dα7/c17 GAL4</i> and<i>PΔEY6/Y; UAS Dα7/c17 GAL4,</i> respectively. The success rate of long-latency stimulation was as follows:<i>PΔL1</i> 8/10,<i>PΔ14G</i> 2/4,<i>PΔ41</i> 0/5,<i>PΔD5</i> 0/6,<i>PΔD5/Y; UAS Dα7/c17 GAL4</i> 2/2,<i>PΔEY5</i> 4/6,<i>PΔEY6</i> 0/4,<i>PΔEY6/Y; UAS Dα7/c17 GAL4</i> 3/3,<i>gfA¹</i> 0/3.</p> <p>(C) Representative traces showing the presence of short- and long-latency responses.<i>PΔEY5</i> and<i>PΔEY6/Y; UAS Dα7/c17 GAL4</i> showed both short- and long-latency TTM responses, but the long-latency response was absent in<i>PΔEY6</i>.</p></div

Inhibition of NLRP3 Inflammasome Pathway by Butyrate Improves Corneal Wound Healing in Corneal Alkali Burn

Epithelial cells are involved in the regulation of innate and adaptive immunity in response to different stresses. The purpose of this study was to investigate if alkali-injured corneal epithelia activate innate immunity through the nucleotide-binding oligomerization domain-containing protein (NOD)-like receptor family pyrin domain containing 3 (NLRP3) inflammasome pathway. A unilateral alkali burn (AB) was created in the central cornea of C57BL/6 mice. Mice received either no topical treatment or topical treatment with sodium butyrate (NaB), β-hydroxybutyric acid (HBA), dexamethasone (Dex), or vehicle (balanced salt solution, BSS) quater in die (QID) for two or five days (d). We evaluated the expression of inflammasome components including NLRP3, apoptosis-associated speck-like protein (ASC), and caspase-1, as well as the downstream cytokine interleukin (IL)-1β. We found elevation of NLRP3 and IL-1β messenger RNA (mRNA) transcripts, as well as levels of inflammasome component proteins in the alkali-injured corneas compared to naïve corneas. Treatment with NLRP3 inhibitors using NaB and HBA preserved corneal clarity and decreased NLRP3, caspase-1, and IL-1β mRNA transcripts, as well as NLRP3 protein expression on post-injury compared to BSS-treated corneas. These findings identified a novel innate immune signaling pathway activated by AB. Blocking the NLRP3 pathway in AB mouse model decreases inflammation, resulting in greater corneal clarity. These results provide a mechanistic basis for optimizing therapeutic intervention in alkali injured eyes