TLR7 Agonists Modulate the Activation of Human Conjunctival Epithelial Cells Induced by IL-1β via the ERK1/2 Signaling Pathway

Conjunctival epithelia cells play an important role in the development of allergic reactions. TLR7 agonists have been shown in studies to increase the body’s immunological tolerance by controlling the proportion of Th1/Th2 cells, although it is still unknown what impact this has on conjunctival epithelial cells. In this study, we examined the effect of TLR7 agonists on the inflammatory-activation of conjunctival epithelial cells induced by IL-1β. Quantitative PCR and ELISA analysis confirmed that TLR7 agonists could impair the proinflammatory cytokines released by the epithelia cells, whereas pro-inflammatory cytokines led to subsequent reactive oxygen species and neutrophil chemotaxis. Phosphorylation analysis and nucleocytoplasmic separation further confirmed that TLR7 agonists inhibit IL-1β-induced epithelia cells activation and ATP depletion via modulating the cytoplasmic residence of ERK1/2. Our finding indicated that TLR7 of conjunctival epithelia cells could be as a potent anti-inflammatory target for the ocular surface. And TLR7 agonists may become potential new drug for the treatment of allergic conjunctivitis.


INTRODUCTION
In allergic conjunctivitis, conjunctival epithelial cells play an important role [1][2][3]. As an impermeable barrier, healthy conjunctival epithelium allows nutrients, small molecules, and water to pass through while blocking pathogens, pollutants, and allergens [4]. In addition to acting as a mechanical barrier, pro-inflammatory cytokines have been shown to activate conjunctival epithelial cells [5]. Inflammatory mediators such as cytokines, adhesion molecules, and chemokines are secreted by activated conjunctival epithelial cells, promoting the infiltration of immune cells (neutrophils, eosinophils) into the site of allergeninduced inflammation in the conjunctiva [1,5]. Inflammation damages the epithelial barrier, resulting in a variety of ocular surface diseases, including allergic conjunctivitis (AC). Tumor necrosis factor α (TNFα), interleukin 1β (IL-1β), intercellular adhesion molecule 1 (ICAM-1), and interleukin-8 (IL-8) expression and TLR7 Agonists Modulate the Activation of Human Conjunctival release were increased when conjunctival epithelial cells were stimulated by pro-inflammatory cytokines.
Interleukin 1 (IL-1) is a critical cytokine in the late-phase allergic response. IL-1β can mediate or amplify virtually every aspect of the pathobiology and pathophysiology of allergic eye disease. Previous research has shown that topical IL-1β inhibition can safely reduce the signs and symptoms of AC and Dry Eye Disease (DED) [6,7]. All of the evidence suggested that IL-1β signaling is the primary cause of conjunctivitis. IL-1β is an upregulator of the release of IL-8 and is elevated in tears from patients with all forms of AC [8,9].
Toll-like receptor (TLR) agonists appear to be safe compounds for the immune-modulatory response toward tolerance to allergy [10,11]. Because of their ability to modulate the T-helper 1 (Th1)/T-helper 2 (Th2) immune response, all TLR7 agonists could be considered as allergy vaccine adjuvants. TLR7 agonists, such as AZD8848, could potentially reduce allergen responsiveness in patients with allergic asthma by stimulating type 1 interferon responses, which downregulate the dominant Th2 responses [12]. Imiquimod and its derivatives have distinct immune-stimulatory effects on both human plasmacyte-like dendritic cells (PDCs) and myeloid dendritic cells (MDCs), promoting the differentiation of naive T cells into Th1 cells via distinct cytokines [11]. Imiquimod and Resiquimod have been studied in vitro and in animal models for AR and asthma [13]. The compounds' ability to reverse Th2 responses in favor of an anti-allergic Th1 response has been demonstrated repeatedly [14].

Cells Culture
Primary conjunctival epithelial cells (CECs) [15] and limbal stem cells (LSCs) [16] were co-cultured using primary epithelial culture basic medium (PECBM, ImmorTech, Shenzhen, China) with feeder cells or cultured in primary epithelial culture medium (PECM, ImmorTech, Shenzhen, China) without feeder cells at 37 ℃ with 5% CO 2 [15,16]. This research followed the tenets of the Declaration of Helsinki. Informed consent was obtained from the subjects after explanation of the nature and possible consequences of the study. The research was approved by the Institutional Review Boards at the Shenzhen Eye Hospital.

Neutrophils Chemotaxis Experiment
LSCs and CECs were plated in 24-wells and merged as monolayers. 2 × 10 5 Neutrophils were added into the insert and co-cultured with LSCs or CECs for 3 h in FBS free medium. Then, inserts were washed with PBS for 3 times and fixed with 4% para-formaldehyde. The chemotaxis of neutrophils on membrane of the insert was stained with crystal violet.

Reactive Oxygen Species Analysis
Wash cells 1 or 2 times with 1 × Buffer. Stain cells by adding diluted 2, 7-dichlorodihydrofluorescein acetoacetic acid (DCFDA) Solution. Incubate cells with the diluted DCFDA Solution for 45 min at 37 °C in the dark. Wash cells 1 or 2 times with 1 × Buffer. Perform live cell microscopy with a filter set appropriate for fluorescein (FITC). The above operations are based on the manual protocol (abcam, ab113851, UK).

ATP Production Analysis
LSCs and CECs were seeded in 24-wells plate for 2 × 10 5 per well and pre-treated with TLR7 agonists (10 µg/ml IMQ, 20 µM AZD8848, 10 µM HY103039) for 2 h, followed by treatment with 10 ng/ml IL-1β for 2 h. Then, the cells were collected and the intracellular ATP was measured by using ATP analysis kit (Beyotime, S0026, China) and BioTek SYNERGY H1 microplate reader according to the manufacturer's instructions.

Statistical Analyses
Averaged data are shown as the mean ± standard error (s.e.) of the mean. Differences in concentrations were analyzed by analysis of variance. If significant differences were found, an appropriate multiple comparison procedure (Student-Newman-Keuls test) was performed. The level of statistical significance was set at P < 0.05. Analyses were performed using GraphPad Prism 7.0 software for Windows.

IL-6, IL-8, and IL-33 Were Induced by IL-1β Stimulation in Human Primary Conjunctiva Epithelia Cells
Interleukin (IL)-1 mediates and amplifies nearly all aspects of ocular allergic disease pathology and pathophysiology. Increased IL-1β expression was found in all types of AC patients [9,17], and increased IL-1βsecretion was found in dry eye disease and other ocular inflammatory diseases [6]. We used IL-1β (10 ng/mL) to stimulate primary human conjunctiva epithelial cells in this study. TLR7 mRNA level was induced by IL-1β periodicity, as shown in Fig. 1a. IL-1β stimulation increased TLR7 protein levels in the cytoplasm after 48 h (Fig. 1b). Increased IL-1β and other inflammatory factor expressions in tears stimulate the conjunctival and corneal epithelium, affect tight junctions between epithelial cells, promotes epithelial cell necrosis, and aggravates inflammatory reactions [18]. As a consequence, we examined the expression and secretion of IL-6, IL-8, and IL-33 by conjunctiva epithelial cells at various time points. Real-time PCR analysis revealed that IL-1β significantly increased the mRNA levels of IL-6, IL-8, and IL-33 from the 2 h time point (Fig. 1c). The levels of IL-6, IL-8, and IL-33 secretion in cell culture supernatants were then measured using an ELISA experiment. The results revealed that the IL-6 and IL-8 sustainability increased in the IL-1β stimulation group when compared to the control group. While compared to the control group, IL-33 in the treated group showed fluctuating secretion and constant upregulation, and was higher at 0 h, 6 h, and 72 h. (Fig. 1d). Furthermore, we found reactive oxygen species (ROS) in IL-1βstimulated conjunctiva epithelial cells. ROS could be seen as early as 48 h, and staining enhanced as processing time was extended (Fig. 1e). The findings show that when stimulated by IL-1β, conjunctival epithelial cells secrete pro-inflammatory cytokines and chemokines, which may affect immune cell chemotaxis. However, the significance of TLR7 in response to IL-1β stimulation remains unknown and requires further investigation.

TLR7 Agonist Suppressed the Expression and Secretion of IL-6, IL-8 and IL-33 Induced by IL-1β
By activating TLR7 on immune cells, TLR7 agonists have been shown to have protective benefits in both OVAinduced asthma models and allergy models in mice [10,19]. However, there are not many publications on TLR7 function in ocular surface epithelial cells during allergic reactions. Three different TLR7 agonists were utilized to preprocess cells 2 h prior to IL-1β stimulation in order to determine whether TLR7 of conjunctiva epithelia cells engage in immune modulatory. After 2 h of IL-1β treatment, cells were extracted, and QPCR was used to examine the expression of IL-6, IL-8, and IL-33. Limbal stem cells served as the control cells. IL-6 expression was significantly reduced in conjunctival epithelia cells and limbal stem cells after IMQ administration, respectively. Similar outcomes were seen in cells treated with HY103039 and AZD8848 (Fig. 2a). The chemokine family includes IL-8, which may draw neutrophils. The findings demonstrated that all TLR7 agonists we utilized diminished the activation of IL-8 by IL-1β (Fig. 2b). As a member of the IL-1 family with high levels of expression in endothelial, epithelial, and fibroblast-like cells, IL-33 plays a critical role in immune regulation with pleiotropic effects in type-2, type-1, and regulatory immunological responses, particularly in allergic inflammation. As a result, we also examined the expression of IL-33 mRNA. TLR7 agonists successfully reduced IL-33 expression, as seen in Fig. 2c. Additionally, at the same time, samples of the IL-6, IL-8, and IL-33 secretion in cell culture conditions were taken and evaluated. TLR7 agonists had no impact on the suppression of IL-6 secretion in LSCs in Fig. 2d. However, there were noticeable decreases in CECs. TLR7 agonists prevented LSCs and CECs from secreting IL-8 (Fig. 2e). However, we did not find a significant inhibitory effect of TLR7 agonists on IL-33 secretion, which could be due to low IL-33 secretion not being easily detected from epithelia cells (Fig. 2f).

Neutrophils Chemotaxis and Reactive Oxygen Species Induced by IL-1β Were Weakened with TLR7 Agonists' Treatment
Although TLR7 agonists suppressed the production and secretion of inflammatory factors, more research is required to determine whether immune cells' chemotaxis may also be impacted. The chemokine interleukin-8 (IL-8) is involved in the recruitment of neutrophils, which develop into a predominate subset of the infiltrate during the pathophysiology of allergies. It is a member of the CXC family of chemokines, which are specific to neutrophils. In this study, cells were monolayer-planted in 24 wells. Cell culture medium was aspirated after pre-treatment with TLR7 agonist in combination with IL-1β stimulation or not, and cells were washed. Then, for 3.5 h with non-FBS media, neutrophils were put into transwell insert on top of monolayer cells. The transwell member was stained with crystal violet to identify the neutrophils. When compared to the control group, the IL-1β stimulation group's neutrophil chemotaxis was increased. Neutrophil chemotaxis was observed to be stronger in TLR7 agonist treatment groups compared to control cells. It is interesting to note that cells pre-treated with TLR7 agonists and subsequently activated by IL-1β had decreased neutrophil chemotaxis (Fig. 3a). were treated with 10 ng/ml IL-1β for 72 h, and the mRNA and b cytoplasmic/nuclear TLR7 were analyzed at different time points. c The mRNA levels of IL-6, IL-8 and IL-33 were analyzed using quantitative PCR. d Cell culture medium was collected, and the secretion levels of IL-6, IL-8 and IL-33 were analyzed using ELISA. e ROS was analyzed using ROS stain kit. The data are expressed as the mean ± S.E.M. of at least three independent experiments. *p < 0.05; **p < 0.001; ***p < 0.001; ****p < 0.0001.

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Reactive oxygen species (ROS) are always stimulated in conjunction with inflammatory cytokines [20]. We exposed cells to TLR7 agonists and IL-1β alone or in combination for 72 h to evaluate the effect of TLR7 agonists on IL-1β generated ROS. We found ROS in LSC and CEC cells that had been activated by IL-1β. Between the IL-1β stimulated group and the IMQ-treated group, there was a substantial difference. IMQ treatment obviously Fig. 2 TLR7 agonists inhibited pro-inflammatory cytokines expression and secretion. Human limbal stem cells (LSCs) and CECs were pretreated with or without the TLR7 agonists (10 µg/ml IMQ, 20 µM AZD8848, 10 µM HY103039) for 2 h, followed by treatment with 10 ng/ml IL-1β for 2 h. The mRNA levels of IL-6 (a), IL-8 (b), and IL-33 (c) were measured in cells collected for quantitative PCR. The culture medium was collected for ELISA, and the levels of IL-6 (d), IL-8 (e), and IL-33 (f) secretion were measured. The data are expressed as the mean ± S.E.M. of at least three independent experiments. *p < 0.05; **p < 0.001; ***p < 0.001; ****p < 0.0001. ◂ Fig. 3 TLR7 agonists impaired IL-1β induced neutrophils chemotaxis and reactive oxygen species (ROS). a LSCs and CECs were pretreated with or without the TLR7 agonists (10 µg/ml IMQ, 20 µM AZD8848, 10 µM HY103039) for 2 h, followed by treatment with 10 ng/ml IL-1β for 2 h. 2 × 10 5 Neutrophils were co-cultured with LSCs or CECs in inserts with FBS free medium for 3 h. The inserts were stained with crystal violet. b LSCs and CECs were pretreated with or without the TLR7 agonists (10 µg/ml IMQ, 20 µM AZD8848, 10 µM HY103039) for 2 h, followed by treatment with 10 ng/ml IL-1β for 72 h. ROS stains were performed and detected using fluorescence microscope. The data are expressed as the mean ± S.E.M. of at least three independent experiments. *p < 0.05; **p < 0.001; ***p < 0.001; ****p < 0.0001. TLR7 Agonists Modulate the Activation of Human Conjunctival reduced the quantity of ROS in the cells compared to the IL-1β stimulated group. Additionally, IL-1β-induced ROS levels in LSCs and CECs were reduced by AZD8848 and HY103039 (Fig. 3b).
According to the results mentioned above, CECs and LSCs behave differently when it comes to secreting inflammatory factors in response to IL-1β stimulation. In our research, we found that three different types of TLR7 agonists might alter neutrophil chemotaxis.

ERK1/2 Responded to TLR7 Agonists' Administration
IL-6, IL-8, and IL-33 were produced by IL-1β stimulation, but were blocked by TLR7 agonists. ERK1/2 responded to the injection of TLR7 agonists (Fig. 4). We want to look into this inhibition's regulatory mechanism. The MAPK kinases are a family of serine/threonine kinases that control intracellular processes like cell proliferation, differentiation, and inflammation, among other things [21]. Furthermore, NF-B, a transcription factor for IL-6 and IL-8, is examined. In CECs, IL-1β stimulation modestly reduced ERK1/2 phosphorylation. As seen in Fig. 4, treatment with IMQ, AZD8848, or HY103039 alone clearly caused the phosphorylation of ERK1/2. In pretreated cells with IMQ, AZD8848, or HY103039 followed by IL-1β, the degree of ERK1/2 phosphorylation was still strongly elevated. On the other hand, neither JNK nor p38's phosphorylation could be seen in LSCs or CECs. Similar outcomes were found when p65 was also recognized as being phosphorylated, as opposed to p38 and JNK. IL-6 and IL-8 transcription has been revealed to be regulated by ERK1/2 in earlier research [22,23]. It is noteworthy to note that in this study, the phosphorylation of ERK1/2 is induced together with the suppression of IL-6 and IL-8.

The Inhibition of IL-6, IL-8, and IL-33 Secretion Did not Depend on Total ERK1/2 Phosphorylation
FR180204 is a selective ERK inhibitor. With FR180204, which inhibits the ERK1/2 signaling cascade, the role of ERK1/2 in the regulation of TLR7 agonists was further investigated. In Fig. 5a, IMQ and the other two TLR7 agonists increased the phosphorylation of ERK1/2. The level of ERK1/2 phosphorylation then dramatically decreased in CECs with the addition of 10 µM FR180204, but not clearly in LSCs. As seen in Fig. 5b, adding FR180204 to IL-1β stimulation groups had no impact on the expression of pro-inflammation cytokines in LSCs. However, in CECs, FR180204 suppressed IL-1β-induced mRNA expression of IL-6, IL-8, and IL-33. There were no observable differences between IMQ and the other two TLR7 agonists' pretreated cells. After that, ELISA analysis was carried out. IL-8 secretion was unaffected by the combined effects of FR180204 (Fig. 5c, upper panel). IL-6 secretion levels in FR180204 treatment groups in CECs were comparable to levels in groups that received no FR180204 treatment, although they were still lower than levels in the IL-1β group (Fig. 5c, lower panel). In the cell supernatant, IL-33 production is too low to be detected. According to the aforementioned findings, TLR7 agonists did not primarily suppress pro-inflammatory cytokines by phosphorylating total ERK1/2.

Neutrophil Chemotaxis and ROS Were Unaffected by Phosphorylation of Total ERK1/2
Pro-inflammatory cytokines induced neutrophil chemotaxis. As seen in Fig. 6a, the neutrophil chemotaxis induced by IL-1 in both LSCs and CECs was suppressed by all three types of TLR7 agonists. FR180204 administration had no discernible effects on neutrophil chemotaxis in either LSCs or CECs. Similar outcomes were seen in the ROS analysis (Fig. 6b). A selective and ATP-competitive ERK inhibitor is FR180204. We Fig. 5 The effects of ERK1/2 phosphorylation inhibitor FR180204 on pro-inflammatory cytokines expression. a LSCs and CECs were pretreated with or without the TLR7 agonists (10 µg/ml IMQ, 20 µM AZD8848, 10 µM HY103039) for 2 h, followed by treatment with 10 ng/ml IL-1β and 10 µM FR180204 for 2 h. Cells were harvested and western blotting was performed to detect the level of p-ERK1/2 and ERK1/2. b Quantitative PCR was performed to detect the expression level of IL-6, IL-8, and IL-33. c Cell culture medium was collected, and the secretion levels of IL-6 and IL-8 were analyzed using ELISA. TLR7 Agonists Modulate the Activation of Human Conjunctival postulated that phosphorylated ERK1/2 is the primary regulator of suppression of pro-inflammatory cytokines release because TLR7 agonists specifically promoted ERK1/2 phosphorylation. However, when cells were treated with FR180204, no reversible reaction was seen. The potential cause is that FR inhibits inflammation in epithelial cells by acting on other targets.

TLR7 Agonists Altered ERK1/2 Translocation in CECs
When ERK1 and ERK2 homodimers are phosphorylated, they can connect with and activate their appropriate cytoplasmic substrates [24]. We used immunoblotting to identify changes in the distribution of ERK1/2 in Fig. 6 The effect of FR180204 on neutrophils chemotaxis and ROS. a LSCs and CECs were pretreated with or without the TLR7 agonists (10 µg/ ml IMQ, 20 µM AZD8848, 10 µM HY103039) for 2 h, followed by treatment with 10 ng/ml IL-1β and 10 µM FR180204 for 2 h. 2 × 105 neutrophils were co-cultured with LSCs or CECs in inserts with FBS free medium for 3 h. The insert were stained with crystal violet. The ratio of neutrophils chemotaxis was measured by Image J software. b ROS stains were performed and detected using fluorescence microscope. The data are expressed as the mean ± S.E.M. of at least three independent experiments. *p < 0.05; **p < 0.001; ***p < 0.001; ****p < 0.0001. the nucleus and cytoplasm in order to further examine whether TLR7 agonists influence the translocation of ERK1/2. As seen in Fig. 7, the groups of CECs treated with TLR7 agonists had higher levels of cytoplasmic phosphorylated ERK1/2. And IL-1 prevented the cytoplasmic phosphorylation of ERK1/2. With or without TLR7 agonists, there was, however, no glaring difference in LSCs. Additionally, FR180204 encouraged the preservation of ERK1/2 in the cytoplasm.

TLR7 Agonists Accelerated Cellular ATP Production and Modulated SOD2 Expression
Cell proliferation and death can both be reduced by ATP depletion. IMQ or the other two TLR7 agonists might prevent the ATP depletion when IL-1β stimulation (Fig. 8a). Furthermore, we check SOD2, a ROS marker, for expression levels. SOD2 expression was markedly increased by IL-1β stimulation in CECs, as seen in Fig. 8b. SOD2 expression levels in CECs treated with IMQ were identical to control values. The IL-1β-induced expression of SOD2 may be inhibited by AZD8848. And the outcomes in the HY1030309 group matched those of the first two TLR7 agonists. These results demonstrated that TLR7 agonists have a significant role in antiinflammatory damage in ocular epithelial cells at low concentrations.

DISCUSSION
As barriers, the conjunctival epithelium and corneal epithelium shield the eye from the outer environment. An allergic reaction frequently results from the epithelial barrier being exposed to environmental stress [25,26]. In our investigation, we mimicked the response of epithelium to inflammatory cytokine stimulation using human primary corneal epithelial cells (LSCs) and human primary conjunctival epithelial cells (CECs) as cell models. We discovered that LSCs and CECs respond to IL-1β stimulation in very different ways. For instance, whereas the quantity of IL-6 secreted by CECs was almost three times that of control cells, the levels of pro-inflammatory substances secreted by LSCs were lower and IL-6 did not significantly change after 2 h of stimulation. As a result of IL-1β stimulation, CECs secrete IL-8 around 20 times more than LSCs do (Fig. 2b). Additionally, LSCs were less susceptible to IL-1β activation than CECs were to intracellular signaling pathways including ERK1/2 phosphorylation.
More research is being done on TLR7 as a potential target for allergy treatment. People administer TLR7 agonists to enhance immunological tolerance, balance the Th1/Th2 ratio, and upregulate IL-12 and IFN-γ while downregulate IL-4 and IL-5 [12].Clinical trials for allergic asthma have included Fig. 7 TLR7 agonists modulated the cytoplasm retention of phosphorylated ERK1/2. Cytoplasmic/nuclear proteins were separated and immunoblotting was performed to analyze the level of ERK1/2 and the phosphorylation of ERK1/2. TLR7 Agonists Modulate the Activation of Human Conjunctival the TLR7 agonist AZD8848 [27]. In this investigation, we discovered that IL-1β stimulation up regulated TLR7, causing both LSCs and CECs to secrete more pro-inflammatory cytokines. The aforementioned phenomena can be seen more vividly in CECs as compared to the lackluster reaction of LSCs. Higher neutrophil counts in the eyes are associated with more severe signs and symptoms. In the corneas of mice, neutrophil extracellular traps lead to epitheliopathy [28]. In the neutrophil recruitment experiment, we also discovered that the recruitment of neutrophils was inhibited after TLR7 agonist pre-treatment of Fig. 8 TLR7 agonists induced ATP production and SOD2 expression. a LSCs and CECs were pretreated with or without the TLR7 agonists (10 µg/ml IMQ, 20 µM AZD8848, 10 µM HY103039) for 2 h, followed by treatment with 10 ng/ml IL-1β and 10 µM FR180204 for 2 h. The intracellular ATP content was analyzed using ATP kits. b SOD2 expression was analyzed by using quantitative PCR. The data are expressed as the mean ± S.E.M. of at least three independent experiments. *p < 0.05; **p < 0.001; ***p < 0.001;****p < 0.0001. cells. It is interesting to note that when TLR7 agonists were administered alone, epithelial cells greatly increased their recruitment of neutrophils compared to the control group, which was consistent with the earlier finding that TLR7 activation could drive an inflammatory response. However, in IL-1β stimulated cells, the ability of cells with TLR7 agonist pretreatment to recruit neutrophils is reduced in comparison to the IL-1β treatment group. These findings suggest that, depending on the context, TLR7 activation has a dual effect on neutrophil recruitment of epithelial cells. Further ROS labeling revealed that cells pretreated with TLR7 agonist had lower ROS levels than the group that had just been activated with IL-1β. The aforementioned findings show how TLR7 agonists can control immune cell chemotaxis and shield epithelial cells from oxidative damage brought on by IL-1β activation.
NF-κB signaling or the MAPK signaling pathways are typically responsible for controlling the production of inflammatory factors [29,30]. As a result, we were able to identify the protein levels and phosphorylation levels of the pathways' major players, ERK1/2, p38, JNK, and p65. After 2 h of IL-1β stimulation, it was discovered that the protein levels and phosphorylation levels of p38, JNK, and p65 remained unchanged. But IMQ and other TLR7 agonists markedly increased ERK1/2 phosphorylation. It was shown that ERK1/2 phosphorylation inhibitor FR180204 was unable to counteract the effect of TLR7 agonists on the release of inflammatory factors, indicating that ERK1/2 phosphorylation may not be the only mechanism that TLR7 agonists function. ERK1/2 plays a varied regulatory role in the cytoplasm and nucleus, and phosphorylation causes ERK1 and ERK2 to form heterodimers, which further regulate the cytoplasmic activity of ERK1/2 [31][32][33][34]. As a consequence, we examined ERK1/2 distribution in the cell further and discovered that TLR7 agonists elevate ERK1/2 phosphorylation levels in the cytoplasm. These findings imply that TLR7 agonists further modified ERK1/2 function in regulating the transcription of inflammatory factors by controlling ERK1/2 translocalization to the nucleus, although further study into the molecular mechanism is still necessary. Additionally, all three of the TLR7 agonists prevented the ATP depletion that IL-1β caused in the cells. However, more research is required to determine whether TLR7 is required for the inhibition of TLR7 agonists on ATP depletion brought on by IL-1β.

CONCLUSIONS
In conclusion, our research revealed that TLR7 agonists limit conjunctival epithelial cell activation by impairing the release of chemokines and ROS brought on by IL-1β through ERK1/2 signaling pathways.
This study presented a possible therapeutic target for the treatment of allergic conjunctivitis as well as a fundamental molecular mechanism by which TLR7 agonists control the course of ocular surface inflammation.

AUTHOR CONTRIBUTION
LW and LY contributed to the conception of the manuscript. LW wrote the manuscript. KH C and SX L draw the pattern diagrams. LW, SX L, and Y X performed experiments. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. All authors read and approved the final manuscript.

DATA AVAILABILITY
The data and materials that support the findings of this study are available from the corresponding author upon reasonable request. TLR7 Agonists Modulate the Activation of Human Conjunctival provide a link to the Creative Commons license, and indicate if changes were made. The images or other thirdparty material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.