The Role Of GSDMD In The Immune Regulation Of PRV Infection-induced Lung Lesions

Pseudorabies virus（PRV）is a pathogen of pig,with a wide range of infections and various infection routes,which not only causes huge economic losses to the global pig industry,but also endangers the life and health of people and animals.As the natural host of PRV,in addition to neurological dysfunction,it also causes pig lung tissue lesions,which subsequently develops into interstitial pneumonia and hemorrhagic pneumonia.Studies show that respiratory diseases are mostly related to immune dysregulation and ncontrolled inflammatory balance,among which immune cells play an important role in regulating the inflammatory response of the body.Gasdermin D（GSDMD）is a key executive molecule in the pyroptosis pathway,which can regulate immune cell death and the release of related inflammatory factors,and has recently been regarded as a novel ideal target for the treatment of inflammatory imbalance type diseases.Pyroptosis is widely involved in the formation of respiratory diseases,but the role of GSDMD in interstitial pneumonia caused by PRV infection is still unknown.In this study,a mouse model of interstitial pneumonia induced by PRV-infected mice was successfully constructed,and GSDMD knockout mice were used to further explore the role of GSDMD in the process of lung tissue lesions caused by PRV infection.The results are shown below:1.Mouse model of interstitial pneumonia with PRV infection was successfully constructedPRV-RJ wild strain and Bartha-K61 vaccine strain were selected for amplification and culture,and their virus titer was determined by Kou Shi（Karber）method.The non-infected group and Bartha-K61 infected group were used as controls to construct PRV infected mouse models with high dose and low dose,respectively.After intraperitoneal injection of high dose(2×10⁵ TCID₅₀)PRV-RJ wild strain,the mice died 48～60 h after infection and showed obvious neurological symptoms,The death time of mice in the Bartha-K61infection group at the same dose was concentrated at 8～10 days after infection.The lungs and brain of mice were examined 48 h after infection,and it was found that the lungs of mice infected with high dose PRV-RJ showed solid changes,accompanied by severe interstitial pneumonia,focal hyperemia of left cerebral meninges,and obvious"vascular sheath"phenomenon.Immunohistochemistry showed that PRV positive signal could be detected in both lung and brain tissues.The lung and eye views of the high-dose Bartha-K61 infected group were roughly the same as those of the normal group,and no obvious brain lesions were observed.After intraperitoneal low-dose injection(2×10³TCID₅₀)PRV-RJ wild strain,the death time of the mice was concentrated at 96 h after infection,the mice showed respiratory symptoms during the infection,but the Bartha-K61infected group at the same dose did not die,and they had almost no obvious clinical symptoms within 96 h of infection.Lung and brain tissues were dissected at 90 h after infection,and it was observed that the lungs of mice infected with low-dose PRV-RJ were fleshy red with typical interstitial pneumonia,while the lungs of mice infected with low-dose Bartha-K61 were slightly swollen.Immunohistochemistry showed that compared with the low-dose Bartha-K61 group,the lung tissues of mice infected with low-dose PRV-RJ showed more obvious PRV positive signal.Furthermore,there were no significant brain lesions in both groups of infected mice.The results showed that high-dose PRV-RJ wild strain infected mice caused significant neurological symptoms,and low-dose PRV-RJ wild strain infected mice mainly induced typical interstitial pneumonia.2.GSDMD deletion can significantly reduce lung lesions caused by PRV infection in miceHigh-dose(2×10⁵ TCID₅₀),low-dose(2×10⁵ TCID₅₀)PRV-RJ wild strain and high-dose(2×10⁵ TCID₅₀),low-dose(2×10³ TCID₅₀)Bartha-K61 vaccine were used for wild-type（WT）mice and GSDMD knockout mice,respectively.To compare the survival,clinical symptom score,viral load,and histopathological changes in WT mice and GSDMD knockout mice in the three models.The results showed that GSDMD deficiency appropriately prolonged survival in mice,alleviated clinical symptoms,and reduced lung and brain tissue lesions in a high-dose PRV-RJ infection model;In the low-dose PRV-RJ infection model,GSDMD deletion prolonged survival and even prevented mice,the clinical symptom scores of GSDMD knockout mice were significantly lower than WT mice,GSDMD deficiency improved lung tissue lesions caused by PRV infection;In a high-dose model infected with the Bartha-K61 vaccine strain,GSDMD deletion extended survival time and reduced lung lesions in mice;In low-dose Bartha-K61 vaccine strain infection model,GSDMD deletion relieved clinical symptoms and lung lesions in infected mice,although no death was observed in WT and GSDMD knockout mice.The above results indicate that GSDMD has different effects in the process of lung tissue lesions caused by different doses and different strains of infected mice.Specifically,GSDMD deficiency can alleviate lung inflammation and pathological damage caused by PRV-RJ wild strain and Bartha-K61 vaccine strain.3.To investigate the regulation of GSDMD on immune cell population during the formation of PRV infection induced interstitial pneumoniaAfter low-dose PRV-RJ wild strain infected WT mice and GSDMD knockout mice,the proportion and number of immune cells,Natural killer cells（NK cells）,Alveolar macrophages（AMs）,Interstitial macrophages（IMs）in the lungs of mice at different infection time points were detected by flow cytometry.The results showed that GSDMD could regulate immune cells,NK cells and macrophages in PRV-infected pig lung tissue.Specifically,the deletion of GSDMD could reduce the level of immune cell infiltration and stabilize the proportion and number of NK cells and macrophages.Meanwhile,single-cell transcriptome sequencing was performed on lung tissues of WT mice and GSDMD knockout mice at the late stage of PRV-RJ infection.The results showed that single cell sequencing divided lung cells into 15 cell subpopulations.Further differential gene analysis of NK cells,AMs and IMs subpopulations in two groups of mice showed that GSDMD deficiency during late PRV infection could upregulate S100a6,Zeb2 and other genes in mouse lung NK cells to maintain NK cell activity and play the role of killing virus;GSDMD deletion can also down-regulate the expression of inflammatory and immune system-related genes（such as Il1b,Tnf,Cxcl3,Ccrl2,etc.）in mouse lung AMs,thereby avoiding excessive inflammatory and immune responses by reducing the over-activation of AMs;In addition,differential genes such as Rnasel、Cxcl16 were up-regulated and differential genes such as Tsc22d3、Areg、Irf7 were down-regulated in mouse lung IMs,suggesting that GSDMD deletion can promote the activation of IMs and enhance the antiviral effect and enhance the anti-inflammatory activity of IMs and prevent excessive inflammation caused by late infection.These results indicate that GSDMD plays an important role in the regulation of NK cells,AMs and IMs during the formation of interstitial pneumonia caused by PRV infection.In conclusion,this study clarified that GSDMD deficiency can significantly reduce the interstitial pneumonia induced by PRV infection.It is further confirmed that GSDMD plays an important regulatory role in the formation of PRV infect-induced interstitial pneumonia by regulating the growth and pro-inflammatory activity of NK cells,AMs and IMs,laying a theoretical foundation for the subsequent study of the specific mechanism,and providing a target for clinical prevention and treatment of pseudorabies.