Effect Of IFN-γ On The Expression Of Two Isoforms Of The ACE2 Gene In HaCaT Cells

Psoriasis is an immune-mediated chronic recurrent inflammatory disease involving multiple organs of the skin,joints and the whole body,and its pathogenesis is affected by a combination of genetic and environmental factors.The pathogenesis of psoriasis is complex and not yet fully defined.Psoriasis is a Th 1-mediated autoimmune disease.In the early study of psoriasis,γinterferon（Interfereron Gamma,IFN-γ）was found to play an important role in the formation of psoriasis-like skin lesions in psoriasis.Several studies have also confirmed the increase of IFN-γin the serum of psoriasis patients.Later,with the gradual deepening of research,it was found that many cytokines,such as tumor necrosis factorα（tumor necrosis factor-α,TNF-α）,interleukin 17（Interleukin-17,IL-17）,and interleukin 23（Interleukin-23,IL-23）,also have important roles in the inflammatory response of psoriasis.These immune cells and cytokines interact with each other,leading to the infiltration of immune cells and excessive proliferation of keratinocytes in psoriatic skin lesions.Novel coronavirus（Severe Acute Respiratory Syndrome Coronavirus 2,SARS-Co V-2）has caused a global pandemic since its discovery in 2019.The virus continues to mutate over time,and now it is more contagious,has more subtle transmission routes,and is more susceptible to people.SARS-Co V-2 binds to the human cell angiotensin converting enzyme（Angiotensin-converting enzyme 2,ACE 2）through the S protein receptor binding domain on the surface of the virus.Under the action of serine protease TMPRSS2,it performs a series of structural reorganization,merges with the human cell membrane,and then infects cells and completes the virus replication.The ACE2 gene is an interferon-stimulated gene,and its expression is regulated by IFN-γ.It has been shown that ACE 2 is regulated by IFN-αand IFN-γin human respiratory tract epithelial cells,upregulating its expression,and has tissue-specific.Psoriasis patients also produce interferon response during inflammation,and some studies have confirmed that IFN-γexpression is up-regulated in the serum of psoriasis patients,and ACE 2 expression also up-regulates in the serum and skin lesions of psoriasis patients^[1].However,there is no relevant experimental data to support whether IFN-γregulates ACE 2 expression in epidermal keratinocytes.We speculate that the expanded inflammatory cascade increases IFN-γexpression,which leads to upregulation of ACE 2,the receptor for SARS-Co V-2,and increased susceptibility to SARS-Co V-2.Additional studies have shown that ACE 2 upregulated by interferon or viral infection is not full-length ACE 2,but a shorter ACE 2 isoform,which is named delta ACE2（d ACE 2）^[2].The d ACE 2 cannot bind to the S protein of SARS-CoV-2 and so does not increase susceptibility to SARS-Co V-2 virus after cells are stimulated by interferon or virus infection.Therefore,to explore whether the upregulation of ACE 2 expression in psoriasis patients is due to regulation by cytokines such as IFN-γ,whether the expression isoform of regulated ACE 2 is full-length ACE 2 or d ACE2,and whether patients with psoriasis have a higher risk of infection during the SARS-Co V-2 epidemic,we performed the following experiments.First,we predicted the regulatory effect of multiple cytokines on the expression of ACE 2 using GEO database analysis,and screened the up-regulation of ACE 2expression regulated by IFN-γ.Then,by bioinformatics analysis of ACE 2 expression sequence,we obtained the gene sequence of ACE 2 and d ACE 2,and predicted the protein conformation of d ACE 2.Next,we used the real-time PCR（Quantitative real-time PCR,q PCR）to detect the m RNA expression levels of full-length ACE 2 and d ACE 2 in Ha Ca T cells stimulated with different concentrations of IFN-γ,and obtained the type of ACE 2 regulated by interferon and its effect relationship with interferon dose.Results:1)Through the analysis of GEO database,it was found that only IFN-γcan promote ACE 2 expression in various cytokines.Full-length ACE 2 also has not the same peptide chain domain length as d ACE 2,with d ACE 2 missing about 50%of the sequence length.2)Full-length ACE 2 and d ACE 2 are the two types stably expressed in Ha Ca T cells,and normally full-length ACE 2 is 2-times more expressed than d ACE 2,occupying the dominant role.3)IFN-γcan induce the upregulation of two variable isoforms in Ha Ca T cells,and both full-length ACE 2 and d ACE 2 are up-regulated under the induction of IFN-γ,with the increase of IFN-γconcentration,while the expression of d ACE 2 does not change with IFN-γconcentration.By experiments,we found that upregulated IFN-γcould induce ACE2 expression in Ha Ca T cells in a concentration-dependent manner.In up-regulated ACE2,full-length ACE2 is 2-fold higher than d ACE 2,and full-length ACE 2 can bind to SARS-Co V-2.The above results can provide important data support for studying the susceptibility of psoriasis patients with SARS and SARS-CoV-2.