Calreticulin Attenuates The Inflammatory Intensity Of Herpes Simplex Keratitis By Inhibiting NLRP3/ASC/caspase-1 Inflammasome Activation

Background:Herpes simplex virus 1（HSV-1）infection of the cornea may lead to herpes simplex keratitis（HSK）,causing corneal tissue damage of varying degrees from the epithelium to the endothelium.It is also the main cause of vision loss and infectious blindness.More than 60%of new infections present as epithelial keratitis,which is spotty,dendritic,or cartographic cloudiness of the corneal epithelium.During this period,HSV-1 replicates extensively in the corneal epithelium and its pathogen-associated molecular patterns（PAMPs）are recognized by the hosts’pattern recognition receptors（PRRs）thus releasing a mass of pro-inflammatory signaling molecules and cytokines.Then inflammatory cells（such as neutrophils,dendritic cells,macrophages,etc.）congregate inducing a protective innate immune response aimed at eliminating HSV-1.In general,most of HSV-1 can be cleared,but overactive inflammatory cell infiltration can cause severe corneal tissue damage and over-activate the downstream adaptive immune response,resulting in corneal stroma opacity,corneal lysis and even perforation.In addition,HSV-1,which is not completely eliminated,can invade along nerve endings and lie in the trigeminal ganglion,causing latent infections.HSV-1 may be reactivated uncontrollably and replicate after immune suppression,fever,ultraviolet exposure,trigeminal ganglion damage,and inappropriate corticosteroid use,and transported from the trigeminal ganglion to the corneal epithelium for mass replication afterwards,which induces a more intense cascade of corneal inflammation,leading to irreversible corneal damage and even permanent blindness.6-8 weeks old female BALB/c mice are often used to replicate the disease model of human HSK.The dynamic mechanism of primary corneal infection in mice is similar to the reactivation of latent HSV-1 from the trigeminal ganglion to the cornea in humans.That is,the first infection of the mouse cornea with HSV-1 induces a cascade of corneal inflammation.In addition to causing HSK,HSV-1 infection can also cause herpes labialis,neonatal viral encephalitis,and even increase the susceptibility and infectivity to the human immunodeficiency virus（HIV）,leading to more serious consequences.Antiviral drugs such as acyclovir and interferon can effectively inhibit the replication of HSV-1,but they cannot eliminate the induced inflammatory response and latent infection.Furthermore,due to the widespread corneal toxic side effects of antiviral drugs and increasing drug resistance,the existing treatment has obvious limitations.In addition,there is no effective vaccine against HSV-1 infection currently,so it is important to study the pathogenesis of HSV-1 and search for new therapeutic targets.calreticulin（CALR）is an endoplasmic reticulum resident protein that regulates Ca²⁺homeostasis,assists protein folding,regulates immune response,and participates in tumorigenesis.CALR is known to be associated with Japanese encephalitis virus and HIV infection,but whether CALR plays a role in HSV-1 infection and its specific mechanism have not been reported.Objective:1.To investigate the abnormal expression of CALR in the mice corneas with HSK and human corneal epithelial cells infected with HSV-1.2.To explore the effect of subconjunctival injection of recombinant CALR on the intensity of inflammation in mice with HSK.3.To explore the mechanism of CALR in the inflammatory response of human corneal epithelial cells infected with HSV-1.Research methods:1.HSV-1 Ma Krae strain was inoculated into the right cornea of6-8 weeks old female BALB/c mice to establish a mouse model of HSK and uninfected control was set up.HSV-1 Ma Krae strain was used to infect immortalized human corneal epithelial cells and the multiplicity of infection（MOI）,the ratio of virions to cell numbers,was 10,to establish the cell model of HSV-1 infection and uninfected control was set up.2.The corneal inflammation of mice was observed with a microscope,and the corneal inflammation of mice was evaluated according to the score standard of 0-4 keratopathy.That is,the score of normal cornea was recorded as"0";Corneal epithelial spotty lesions were scored as"1";Dendritic lesions less than 1/4 of the whole cornea were scored as"2";Severe dendritic lesions more than 1/4 of the whole cornea were scored as"3",and cartographic corneal ulcers were scored as"4".The pathological changes of corneal tissue in mice were studied by corneal histopathology.Western Blot,q PCR and immunofluorescence were used to detect the expression and localization of CALR in mouse corneas and cultured immortalized human corneal epithelial cells.3.Recombinant CALR（experimental group）or PBS（control group）was injected subconjunctively to mice 1 day before and 1 and 2 days after HSV-1 infection,and the influence of CALR or PBS on the intensity of corneal inflammation in the two groups was observed with a microscope and the keratopathy score was performed.Corneal histopathology was used to study the effect of CALR or PBS on the pathological changes of corneal tissue of mice in the two groups.The expression of inflammatory factors was studied by Western Blot.4.Cultured immortalized human corneal epithelial cells were transfected with CALR overexpression plasmid（experimental group）or empty vector control group（control group）,and the efficiency of CALR overexpression was verified.After successful transfection of the plasmid,HSV-1 infected cell models were established and cells were collected for m RNA sequencing and biogenic analysis to predict the downstream target genes of CALR.5.After overexpression plasmid or small interfering RNA（si RNA）was used to overexpress or silence CALR in cultured immortalized human corneal epithelial cells,the efficiency of CALR overexpressing or silencing was verified.After successful overexpressing or silencing CALR,a cell model of HSV-1 infection was established,and the influence of overexpressing or silencing CALR on the expression or secretion of predicted target genes and downstream inflammatory factors was studied by Western Blot,q PCR and ELISA.6.The overexpression plasmid was used to overexpress CALR in cultured immortalized human corneal epithelial cells and empty vector control was set up to establish the cell model of HSV-1 infection.Fluo-3 AM calcium probe was used to detect the fluorescence intensity of intracellular free Ca²⁺and explore the regulation of CALR on the concentration of intracellular free Ca²⁺.7.Statistical analysis:The Graghpad prism 8.0 software was used for statistical analysis in this study.T-test was used for comparison between two groups,and one-way analysis of variance was used for comparison between multiple groups.In this study,Graghpad prism 8.0 software was used to draw statistical figures,and Image J software was used to analyze the band gray of Western Blot and cell fluorescence intensity.All data was presented by mean±standard deviation（`x±s）and P<0.05 represented statistically significant.Results:1.In this study,a mouse model of HSK and a cell model of immortalized human corneal epithelial cells cultured with HSV-1 infection were successfully established.2.The corneal inflammation intensity of mice was observed and the corneal pathological changes were studied:（1）The corneal epithelium of mice in the uninfected control group was smooth and transparent.HE staining showed that corneal epithelium was intact,and corneal epithelial cells were arranged regularly,and no inflammatory cells were found in the epithelium and stroma.（2）1 day after inoculation with HSV-1,the corneal epithelium of mice was rough and local epithelium was absent.HE staining showed local deletion of corneal epithelial cells,disordered cell arrangement,and no inflammatory cell infiltration.（3）3 days after inoculation with HSV-1,the corneas of mice were obviously cloudy.HE staining showed that the corneal tissue was thickened,the epithelial cells were arranged disordered,presenting obvious vacuole-like changes,and obvious inflammatory cell infiltration was observed in the epithelium and stroma.（4）5 days after inoculation with HSV-1,the corneal opacity of mice was relieved.HE staining showed that the corneal epithelium was slightly thickened,the intraepithelial inflammatory cells subsided,and no obvious inflammatory cell infiltration was observed in the stroma.Statistical analysis showed that the keratopathy score at different time points were statistically significant（F=19.38,P=0.000）.After paired comparison,it was found that the keratopathy score at 3 days was higher than that at 1day and 5 days after infection with HSV-1（P=0.000,0.000）.Western Blot and q PCR were used to detect the expression of CALR in the corneas of mice.The results showed that the expression of CALR in the corneas of mice with herpes simplex keratitis decreased compared with those without corneas infection（t=4.467,P=0.002 and t=4.062,P=0.002,respectively）.Immunofluorescence showed that CALR was mainly localized in the cytoplasm and nucleus of human corneal epithelial cells,and the fluorescence intensity of CALR decreased in HSV-1 infected cells compared with uninfected cells（t=2.816,P=0.048）.3.The expression of CALR in the cornea of mice was detected by Western Blot after the subconjunctival injection of recombinant CALR or PBS.The results showed that,compared with the control group injected with PBS,the expression of CALR increased after injection of CALR,and this effect maintained at least 3 days after HSV-1 infection（t=3.673,P=0.021）.The keratopathy score and corneal histopathology showed that,compared with the control group injected with PBS,the inflammation intensity of HSK alleviated,keratopathy score reduced and Interleukin-1β（IL-1β）and IL-18 expression decreased（t=2.813,P=0.031 and t=3.223,P=0.023）after injected with recombinant CALR reduced（t=3.920,P=0.017）.4.In this study,CALR was successfully overexpressed in cultured immortalized human corneal epithelial cells and a cell model of HSV-1 infection was established.A series of downstream target genes that may be regulated by CALR gene were successfully predicted by m RNA sequencing and bioinformatics analysis.PYCARD（ASC）gene and its related NLRP3/ASC/caspase-1 inflammasome pathway were successfully screened.5.In this study,CALR was successfully overexpressed and silenced in cultured immortalized human corneal epithelial cells,respectively,and HSV-1 infected cell models were established.Western Blot was used to detect the efficiency of CALR overexpressing and silencing,and the results showed that CALR was successfully overexpressed and silenced（F=36.12,P=0.001 and F=19.62,P=0.002,respectively）.Western Blot and q PCR were used to further detect the expressions of downstream target genes NLRP3,ASC and caspase-1.The results showed that after overexpression of CALR,the expression of inflammasome components NLRP3,ASC,cleaved caspase-1（the active form of caspase-1）,and downstream proinflammatory cytokines IL-1βand IL-18 decreased（all P<0.05）.In contrast,after CALR silenced,the expression of inflammasome components NLRP3,ASC,cleaved caspase-1,IL-1β,and IL-18 increased（all P<0.05）.ELISA was used to detect the secretion of IL-1βand IL-18 in the supernatant of cells.The results showed that the secretion of IL-1βand IL-18decreased after overexpressing CALR,and increased after silencing CALR（all P<0.05）.6.Fluo-3 AM calcium ion probe was used to detect the fluorescence intensity of intracellular free Ca²⁺.The results showed that the fluorescence intensity of free Ca²⁺in the cytoplasm of the normal control group,the empty vector control group and the overexpressed CALR group were different（F=12.79,P=0.002）.For paired comparison,the fluorescence intensity of intracellular free Ca²⁺in normal control group was lower than that in empty vector control group（P=0.002）,and the fluorescence intensity of Ca²⁺in overexpressing CALR group was lower than that in empty vector control group（P=0.031）.Conclusions:1.After HSV-1 infection,CALR expression in mouse corneal and cultured immortalized human corneal epithelial cells is decreased.2.Subconjunctival injection of recombinant CALR alleviated the intensity of inflammation in mice with herpes simplex keratitis.3.CALR alleviated the inflammatory response induced after HSV-1 infection by inhibiting the activation of NLRP3/ASC/caspase-1 inflammasomes and reducing the secretion of IL-1βand IL-18 through reducing the concentration of free Ca²⁺in cultured immortalized human corneal epithelial cells.