1. Developmental Biology
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Visually induced changes in cytokine production in the chick choroid

  1. Jody A Summers  Is a corresponding author
  2. Elizabeth Martinez
  1. Department of Cell Biology, University of Oklahoma Health Sciences Center, United States
Research Article
Cite this article as: eLife 2021;10:e70608 doi: 10.7554/eLife.70608
10 figures, 1 table and 2 additional files

Figures

Figure 1 with 2 supplements
Immunohistochemical localization of IL-6 in chick choroids.

(A, B) Il-6 was localized in treated and contralateral control eyes after 24 hr of recovery from induced myopia (green labeling). (C) Preabsorption of anti-IL-6 with a tenfold molar excess of recombinant chicken IL-6 (1.67 μM) before use on tissue sections abolished IL-6 labeling. Bar=20 µm in (A–C). Choroidal blood vessels are indicated by asterisks (*). Vascular endothelium is indicated by arrowheads (↑). IL-6, interleukin-6 ; LL, lymphatic lacunae; RPE, retinal pigmented epithelium; S, extravascular choroidal stroma.

Figure 1—figure supplement 1
Immunolocalization of IL-6 in the chick choroid.

(A, B) Adjacent longitudinal sections (10 μm thick) of control chick choroids following standard H&E staining (A), and immunolabeling for IL-6 (B). (C, D) Adjacent oblique sections through chick RPE, choroid, and sclera processed for standard H&E staining (C), and immunolabeling for IL-6 (D). Bar=40 µm in (A–D). Choroidal stroma is indicated by asterisks in (A, B) (*). Vascular endothelium is indicated by arrowheads in (B) (↑). BV, blood vessel; IL-6, interleukin-6; RPE, retinal pigmented epithelium.

Figure 1—figure supplement 2
Improved visualization of DAPI labeling in chick RPE and choroid.

DAPI-labeled nuclei in RPE and choroid from Figure 1 were visualized by increasing the DAPI channel (405 nm) intensity. RPE nuclear layer is located between dotted lines in (A–C). IL-6 immunolabeling is visualized with Alexa Fluor 488 (green labeling). RPE, retinal pigmented epithelium.

Figure 2 with 1 supplement
Microarray identifies IL-6 as a gene highly overexpressed in early recovery.

A volcano plot of Affymetrix chicken microarray data indicated that 207 genes were found to be significantly differentially expressed by ≥2-fold in recovering choroids as compared with choroids from normal untreated chicks (p≤0.05). The horizontal dashed red line indicates where p=0.05, with points above the line having p<0.05 and points below the line having p>0.05. The area between the dashed purple lines indicates points having a fold-change less than |2|. IL-6 was increased by 10.83-fold in recovering choroids compared with normal choroids (n=5 birds in each group); p=0.00084, one-way ANOVA using Method of Moments. IL-6, interleukin-6.

Figure 2—figure supplement 1
Refractions of form-deprived (FD; right eyes) and untreated contralateral control eyes (left eyes) following 10 days of form deprivation.

Monocular form-deprivation of chicks in this study resulted in a large negative shift in refractive error. Data are represented as mean ± standard errors. *p=0.0313 Wilcoxon matched-pairs signed-rank test for n=6 birds.

Figure 2—figure supplement 1—source data 1

Refractions of form-deprived and untreated contralateral control eyes.

https://cdn.elifesciences.org/articles/70608/elife-70608-fig2-figsupp1-data1-v2.xlsx
Figure 3 with 1 supplement
Cytokine gene and protein expression in chick choroids.

(A) IL-6 mRNA expression in choroids from control and treated eyes, following 10 days of form deprivation (0 hr/10 days FD), 0.75 hr to 8 days of recovery from form deprivation, normal, untreated eyes (normal), and in eyes recovered for 6 hr, but kept in total darkness (6 hr in dark) (n=5–16 birds in each group) ***p<0.001, **p<0.01, *p=0.013, Wilcoxon signed-rank test for matched pairs. (B) IL-6 protein production by control and recovering choroids following 6 and 24 hr of recovery from induced myopia. Data are expressed as mean ± SEM (n=16) **p=0.0102, paired t-test. (C) Quantification of other proinflammatory cytokines in chick choroids. Gene expression of Interferon gamma (IFNG), interleukin-1B (IL-1B), and tumor necrosis factor alpha (TNF-α) was quantified in control and treated chick choroids following 6 hr of recovery. Additionally, IL-1B mRNA was quantified following 1.5 and 3 hr of recovery. The dashed line indicates the average IL-6 expression in 6 hr recovering choroids (n=6–11 birds in each group) **p=0.0059, Wilcoxon signed-rank test for matched pairs. IL-6, interleukin-6.

Figure 3—source data 1

Cytokine gene and protein expression in chick choroids.

https://cdn.elifesciences.org/articles/70608/elife-70608-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
IL-6 has no direct effect on scleral proteoglycan synthesis.

Incubation of chicken sclera in organ culture with recombinant chicken IL-6 (10 ng/ml) for 24 hr had no significant effect on scleral proteoglycan synthesis (p=0.1439, Student’s t-test, n=11 sclera in each group). IL-6, interleukin-6.

Figure 3—figure supplement 1—source data 1

IL-6 on scleral proteoglycan synthesis.

https://cdn.elifesciences.org/articles/70608/elife-70608-fig3-figsupp1-data1-v2.xlsx
Figure 4 with 1 supplement
Effect of light intensity on IL-6 mRNA expression.

Normal chicks were housed in complete darkness (dark), white LED light of varying intensities (‘low,’ 5 lux; ‘medium,’ 700 lux; ‘high,’ 3150 lux), red LED light (‘red,’ 58 lux), or blue LED light (‘blue,’ 111 lux) for 6 hr at which time choroids were isolated with Il-6 mRNA was quantified by TaqMan real-time PCR (n=6–8 birds [12–16 choroids]) in each group. ***p<0.001, **p<0.01, Kruskal-Wallis test with Dunn’s multiple comparisons. IL-6, interleukin-6.

Figure 4—source data 1

Results of Taqman (IL-6/GAPDH ∆∆C(t)) Light Intensity Experiment.

https://cdn.elifesciences.org/articles/70608/elife-70608-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Spectra of LED light sources as a function of wavelength.
Figure 4—figure supplement 1—source data 1

Spectra of LED light sources as a function of wavelength.

https://cdn.elifesciences.org/articles/70608/elife-70608-fig4-figsupp1-data1-v2.xlsx
Effect of imposed defocus on choroidal IL-6 gene expression.

(A). Spectacle lenses [minus 15 D (–15) or plus 15 D (+15)] were applied to the right eyes of chicks for 6–24 hr. (B). Schematic diagram illustrating the effects of imposed optical defocus on the location of ocular images of distant objects for an emmetropic eye (center); positive lenses move the image plane in front the retina, imposing myopic defocus (left), while negative lenses move the image plane behind the retina, imposing hyperopic defocus (right). (C) Refractive status of chick eyes while wearing –15 D and +15 D lenses. Application of –15 D lenses results in a hyperopic shift in the refraction of normal chick eyes, relative to untreated (no lens) eyes, whereas application of +15 D lenses results in a myopic shift in refraction, compared to untreated eyes. ***p<0.0001, ANOVA with Bonferroni correction for n=2 chicks in each group (five measurements/chick). (D) IL-6 mRNA expression in choroids from control and treated eyes, following 6 or 24 hr of plus lens wear (n=6 and n=27, respectively), 24 hr of minus lens wear (n=34), and normal untreated choroids (n=8). ***p=0.0003, Wilcoxon signed-rank test for matched pairs. (E) Scleral proteoglycan synthesis following 24 hr of lens wear. Proteoglycan synthesis was significantly reduced following 24 hr of + 15 D lens wear, compared to untreated contralateral control eyes (***p=0.00047, paired t-test, n=10) and was significantly increased following 24 hr of –15 D lens wear, compared with untreated contralateral control eyes (*p=0.0403, paired t-test, n=13).

Figure 5—source data 1

Effect of imposed defocus on choroidal IL-6 gene expression.

https://cdn.elifesciences.org/articles/70608/elife-70608-fig5-data1-v2.xlsx
L-NAME inhibits choroidal IL-6 transcription and recovery.

(A) Intravitreal injection of L-NAME (16.2 μmol/eye) immediately prior to recovery significantly reduced IL-6 mRNA levels compared to recovering eyes receiving vehicle only (0.9% NaCl) (**p=0.007, Mann-Whitney U-test, n=7; *p=0.015, Wilcoxon signed-rank test for matched pairs, n=7). (B) L-NAME disinhibits scleral proteoglycan synthesis in recovering eyes. Following 12 hr of recovery from 10 days of form deprivation (FD), scleral proteoglycan synthesis decreased to control levels in vehicle-treated eyes, but remains significantly increased over control levels in L-NAME treated eyes (***p<0.0001, paired t-test, n=16; **p=0.0121 Wilcoxon signed-rank test for matched pairs, n=17; *p=0.0421, Mann-Whitney U-test, n=17). IL-6, interleukin-6.

Figure 6—source data 1

The effect of L-NAME on choroidal IL-6 transcription and recovery.

https://cdn.elifesciences.org/articles/70608/elife-70608-fig6-data1-v2.xlsx
The NO donor, PAPA-NONOate, stimulates choroidal IL-6 production.

Choroids were isolated from normal chicken eyes and were incubated with the indicated concentrations of PAPA-NONOate for 24 hr. (A) IL-6 gene expression was significantly increased in choroids following incubation in 1.5 mM PAPA-NONOate (*p=0.0079, Mann-Whitney U-test, n=4–5 choroids in each group). (B) IL-6 protein concentrations were significantly increased in choroid culture supernatants following incubation with 3–5 mM PAPA-NONOate (**p=0.0079, *p=0.0357, Mann-Whitney U-test, n=3–5 choroids in each group). (C) Incubation of chicken choroids with PAPA-NONOate (1.5 mM) together with the p38 MAPK inhibitor SB203580 (10 μM) abolished the PAPA-NONOate-induced increase in IL-6 mRNA (**p=0.0032, *p=0.0308, Student’s t-test, n=10 choroids in each group). IL-6, interleukin-6.

Figure 7—source data 1

The effect of the NO donor, PAPA-NONOate on choroidal IL-6 production.

https://cdn.elifesciences.org/articles/70608/elife-70608-fig7-data1-v2.xlsx
L-arginine (L-arg), the NOS substrate, stimulates choroidal IL-6 trasncription.

Choroids were isolated from normal chicken eyes and were incubated with the indicated concentrations of L-arg for 24 hr. KCl (50 mM) was added to some cultures to depolarize cell membranes. (A) IL-6 gene expression was significantly increased in choroids following incubation in 5 mM L-arg in the presence of 50 mM KCl (*p=0.0188, Mann-Whitney test, n=9 choroids in each group). (B) Dose response for the effect of L-arg on IL-6 gene expression. IL-6 gene expression was significantly increased in choroids following incubation in 5 mM L-arginine in the presence of 50 mM KCl (*p=0.04, Mann-Whitney test, n=9 choroids in each group). IL-6, interleukin-6.

Figure 8—source data 1

The effect of L-arginine on choroidal IL-6 transcription.

https://cdn.elifesciences.org/articles/70608/elife-70608-fig8-data1-v2.xlsx
Atropine stimulates choroidal IL-6 gene expression.

(A) Intravitreal injection of atropine (240 nmol/eye) into chick eyes following 14 days of form deprivation (myopic eyes) increased IL-6 mRNA levels compared to myopic eyes receiving vehicle only (PBS) (*p=0.0498, Mann-Whitney U-test, n=18). (B) Incubation of chicken choroids in organ culture with 0.15 atropine for 24 hr significantly increased choroidal IL-6 gene expression (**p=0.0092, Student’s t-test, n=16). (C) IL-6 protein concentrations were significantly increased in choroid culture supernatants following incubation with 0.1% atropine (*p=0.0491, Mann-Whitney U-test, n=10 choroids in each group). IL-6, interleukin-6.

Figure 9—source data 1

The effect of atropine on choroidal IL-6 gene expression.

https://cdn.elifesciences.org/articles/70608/elife-70608-fig9-data1-v2.xlsx
Proposed role of IL-6 in the retina-to-sclera signaling cascade.

Myopic defocus initiates a series of signaling events in the retina potentially involving dopamine, nitric oxide synthase (NOS), and nitric oxide (NO) (as well as other mediators not shown). Nitric oxide, synthesized in the retina and/or in the choroid by NOS from L-arginine (L-arg), stimulates choroidal expression of IL-6 via a p38 MAPK-dependent mechanism. Choroidal IL-6, in turn, potentially coordinates many of the features of the choroidal response to myopic defocus including: (1) increased synthesis of hyaluronan synthase 2 (HAS2) and hyaluronic acid (HA), (2) increased synthesis of vascular endothelial growth factor (VEGF), and (3) increased cell proliferation, which result in choroidal thickening, increased vascular permeability, and increased retinaldehyde dehydrogenase 2 (RALDH2), respectively. These choroidal changes lead to the production of scleral growth regulators, such as all-trans-retinoic acid (atRA), that regulate scleral remodeling, such as decreased scleral proteoglycan (PG) synthesis (in chicks) to result in a slowing of ocular growth and recovery from myopia.

Tables

Key resources table
Reagent type(species) or resourceDesignationSource or referenceIdentifiersAdditional information
AntibodyAnti-chick IL-6(Rabbit polyclonal)Bio-RadLaboratoriesCat#: AHP942ZRRID: AB_2127753IF (1:20)
Sequence-based reagentChicken IL-6Taqman GeneExpression AssayThermo FisherScientificGg03337980_m1unlabeled PCR primers andFAM-labeled TaqMan probe
Sequence-based reagentChicken interferon γTaqMan GeneExpression AssayThermoFisherScientificGg03348618_m1unlabeled PCR primers andFAM-labeled TaqMan probe
Sequence-based reagentChicken IL-1βTaqMan GeneExpression AssayThermo FisherScientificGg03347154_g1unlabeled PCR primers andFAM-labeled TaqMan probe
Sequence-based reagentChicken TNF-α(LITAF) TaqMan GeneExpression AssayThermo FisherScientificGg03364359_m1 unlabeled PCR primers andFAM-labeled TaqMan probe
Sequence-based reagentChicken GAPDHTaqMan GeneExpression AssayThermo FisherScientificGg03346982_m1unlabeled PCR primers andFAM-labeled TaqMan probe
Peptide, recombinant proteinChicken IL-6Bio-RadLaboratoriesCat#: PAP003
Commercial assay or kitHigh capacityRNA-to-cDNA kitAppliedBiosystemsCat#: 4388950
Commercial assay or kitDNase treatment& removal kitInvitrogenCat#: AM1906
Chemical compound, drugSB 203580Sigma-AldrichCat#: S8307
Chemical compound, drugAtropine(Sulfate Salt)Sigma-AldrichCat#: A-0257
ChemicalCompound, drugL-NAMESigma-AldrichCat#: N5751
Chemical compound, drugPAPA-NONOateCaymanChemicalCat#: 82,140
OtherDAPI stainInvitrogenCat#: D3571(5 µg/ml)

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