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. 2023 Aug 1;64(11):3.
doi: 10.1167/iovs.64.11.3.

Thymosin β4 Alleviates Autoimmune Dacryoadenitis via Suppressing Th17 Cell Response

Xiaoyu Zhao  1 , Na Li  1 , Ning Yang  1 , Baoyue Mi  1 , Weiyu Dang  1 , Deming Sun  2 , Shanshan Ma  3 , Hong Nian  1 , Ruihua Wei  1
Affiliations

Thymosin β4 Alleviates Autoimmune Dacryoadenitis via Suppressing Th17 Cell Response

Xiaoyu Zhao et al. Invest Ophthalmol Vis Sci. .

Abstract

Purpose: We investigated the therapeutic effect of recombinant thymosin β4 (rTβ4) on rabbit autoimmune dacryoadenitis, an animal model of SS dry eye, and explore its mechanisms.

Methods: Rabbits were treated topically with rTβ4 or PBS solution after disease onset for 28 days, and clinical scores were determined by assessing tear secretion, break-up time, fluorescein, hematoxylin and eosin staining, and periodic acid-Schiff. The expression of inflammatory mediators in the lacrimal glands were measured by real-time PCR. The expression of T helper 17 (Th17) cell-related transcription factors and cytokines were detected by real-time PCR and Western blotting. The molecular mechanism underlying the effects of rTβ4 on Th17 cell responses was investigated by Western blotting.

Results: Topical administration of rTβ4 after disease onset efficiently ameliorated the ocular surface inflammation and relieved the clinical symptoms. Further analysis revealed that rTβ4 treatment significantly inhibited the expression of Th17-related genes (RORC, IL-17A, IL-17F, IL-1R1, IL-23R, and granulocyte-macrophage colony-stimulating factor) and IL-17 protein in lacrimal glands, and meanwhile decreased the inflammatory mediators expression. Mechanistically, we demonstrated that rTβ4 repressed the phosphorylation of signal transducer and activator of transcription 3 (STAT3) both in vivo and in vitro. Activation of the STAT3 signal pathway by Colivelin partly reversed the suppressive effects of rTβ4 on IL-17 expression in vitro.

Conclusions: rTβ4 could alleviate ongoing autoimmune dacryoadenitis in rabbits, probably by suppressing Th17 response via partly affecting the STAT3 pathway. These data may provide a new insight into the therapeutic effect and mechanism of rTβ4 in dry eye associated with Sjögren's syndrome.

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Conflict of interest statement

Disclosure: X. Zhao, None; N. Li, None; N. Yang, None; B. Mi, None; W. Dang, None; D. Sun, None; S. Ma, None; H. Nian, None; R. Wei, None

Figures

Figure 1.
Figure 1.
rTβ4 reduced clinical signs of rabbit autoimmune dacryoadenitis (n = 6). (A) Schema of 1000 µg/mL rTβ4 treatment in rabbit autoimmune dacryoadenitis. (B) Tear production was measured using Schirmer's test before and after 1000 µg/mL rTβ4 administration. (C) Tear BUT was measured using slit-lamp examination to evaluate the tear stability. (D and E) Corneal fluorescein staining was imaged by a slit lamp biomicroscope and scores were measured. *P < 0.05, **P < 0.01, ****P < 0.0001 vs. the corresponding value in the PBS group. ##P < 0.01, ###P < 0.001, ####P < 0.0001 vs. the corresponding value in the model group. The statistical significance of corneal fluorescein staining scores, Schirmer's test, and tear BUT were determined by two-way ANOVA.
Figure 2.
Figure 2.
rTβ4 treatment suppressed inflammatory cell infiltration in LG and conjunctiva and increased conjunctival goblet cell density (n = 6). (A, B) Hematoxylin and eosin–stained photographs of LGs and conjunctivas were represented. Arrow indicates local lymphocytic foci (>50 infiltrating lymphocytes) around vascular or ductal. (C) Conjunctival goblet cell density (mean ± SD) as detected by enumerating filled goblet cells in periodic acid-Schiff (PAS)-stained histological sections of conjunctiva. (D, E) Numbers of lymphocytic foci per 4 mm2 in LGs and conjunctivas were evaluated. (F) Numbers of filled goblet cells per 4 mm2 in conjunctivas were evaluated (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001. The statistical significance was determined by one-way ANOVA.
Figure 3.
Figure 3.
Administration of 1000 µg/mL of rTβ4 regulated Th17 in vivo. Rabbits were sacrificed at the end of treatment and LGs were collected. Tissues were subjected to qRT-PCR or Western blot analysis. (A) mRNA expression of Th17-related genes (RORC, IL-17A, IL-17F, IL-23R, IL-1R1, GM-CSF) were analyzed by qRT-PCR. (B) The protein level of IL-17 expression was analyzed by Western blot assay. And relative protein ratio of IL-17 to β-actin was quantified. Data were representative of three independent experiments (n = 3 rabbits per group in each experiment) and bar graphs indicated mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 4.
Figure 4.
In vivo administration of 1000 µg/mL rTβ4 decreased proinflammatory cytokines. Rabbits were sacrificed at the end of treatment and LGs were collected. Tissues were subjected to qRT-PCR and Western blot. (A) Gene expression profiles of inflammatory mediators (IL-1β, IL-6, IL-23, TNF-α, matrix metalloproteinase (MMP)-9, MMP-2, CXCL-2, CXCL-8, and CCL-20). (B) The protein levels of IL-6 and IL-1β were measured and quantitatively analyzed by Western blot. The values were expressed as mean ± SD. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data were representative of at least three independent experiments.
Figure 5.
Figure 5.
rTβ4 inhibited Th17 immune response in vitro. (A) Treatment with 100 ng/mL rTβ4 suppressed Th17 immune response in vitro. PBLs isolated from diseased rabbits were cocultured with irradiated pLGECs in the presence or absence of rTβ4 for 72 hours. Gene expression of RORC, IL-17A, IL-17F, GM-CSF, IL-23R, and IL-1R1 were analyzed by qRT-PCR. (B) The protein level of IL-17 expression was analyzed by Western blot assay. The relative protein ratio of IL-17 to β-actin was quantified. Data were representative of three independent experiments (n = 3), and bar graphs showed mean ± SD. *P < 0.05, ****P < 0.0001.
Figure 6.
Figure 6.
rTβ4 inhibited Th17 immune response via suppressing STAT3 phosphorylation. (A) Administration of 1000 µg/mL rTβ4 suppressed the protein level of phosphorylated STAT3 in vivo. (B) PBLs induced by pLGECs were pretreated with the STAT3 activator (Colivelin) for 1 hour and coincubated with rTβ4 for 72 hours. The protein expression of IL-17 and pSTAT3 was measured and quantitatively analyzed by Western blot. Data were representative of three independent experiments (n = 3), and bar graphs showed mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.
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