| Systemic lupus erythematosus(SLE)is a multi-organ autoimmune disease characterized by abnormal activation of immune cells and the generation of large amounts of autoantibodies in the body.The disease mainly affects women of childbearing age and can involve organs such as the skin,joints,blood,kidneys,and brain.Staphylococcus aureus(SA)is a common symbiotic bacterium in human microecology,which is mostly colonized in the upper respiratory tract and skin.As a complex opportunistic pathogen,the imbalance of SA in nasal mucosa microecology has been confirmed to be closely related to the progression of SLE.The toxicity factors related to SA have been reported in many studies on different lupus mouse models.The toxicity factors related to SA have been reported in many studies on different lupus mouse models.Studies have shown that SA infection can induce the proliferation and differentiation of polyclonal B cells in the body.On the one hand,SA-associated antigens with molecular mimetic effects,such as SA peptidoglycan(Staphylococcus aureus peptidoglycan,PGN),can induce proliferation and differentiation of mouse B cells with an increase in C57BL/6 and MRL/lpr serum Anti-ds DNA Ig G titers through molecular mimetic effects;on the other hand,other SA-related antigens,especially Staphylococcus protein A(Sp A),can induce B cell polyclonal proliferation and differentiation,thus increasing serum non-protective antibody titers while decreasing serum levels of protective antibodies,thus enabling immune escape of SA and its virulence factors.In addition,some superantigens can induce specific B cell clones to undergo apoptosis.Studies targeting SA-associated T cells have shown that chronic low-dose subcutaneous injection of Staphylococcal enterotoxin B(SEB)of HLA-DQ8 transgenic mice results in expansion of TCR Vβ8+ T cells and significantly elevated levels of anti-ds DNA and anti-Sm antibodies.Kidneys from mice with SEB also showed deposition of Ig G and C3 complement in the glomeruli and multi-organ inflammatory damage.In another experiment,MRL/lpr injected with SEB intravenously every two weeks significantly reduced the number of TCR Vβ8 CD8+ T and CD4-CD8-cells,accompanied by significant decreases in serum Anti-ds DNA Ig G and urinary protein levels,and alleviation of MRL/lpr disease progression.In addition,SA infection can induce differentiation of Th17 cells that are known to promote SLE progression.The above results indicate that even a single SA secretion toxicity factor can have different effects on lupus model disease progression depending on its administration method,dose and administration interval.SA,as a complex opportunistic pathogen,may have different effects on T and B cells depending on the site and mode of infection.Our previous research found that there was an increase SA colonization in SLE skin lesions,and other studies have further confirmed that abnormal elevation of type I interferon levels and neutrophil extracellular traps dysfunction at SLE skin lesions are conducive to abnormal increase in SA colonization.However,it is not yet clear how the abnormal increase in SA at skin lesions affects the progression of SLE.Tissue-resident memory cells(Trm)are a subtype of cells that can reside in tissues for a long time without participating in blood and lymphatic circulation.They can respond rapidly to re-infection with the same microorganisms,accelerating clearance of pathogens.Previous studies have confirmed that increased and abnormally activated CD4+Trm play an important role in inflammation and tissue damage in autoimmune diseases.Our previous research showed that compared with healthy skin,there was a significant increase in CD4+ Trm cell at SLE skin lesions,besides immune response-related genes in CD4+ Trm cell from SLE skin lesions were significantly upregulated.At the same time,other studies have shown that superficial skin colonization of SA can induce Th17-dominated CD4+ T cell infiltration.Therefore,we speculate that the abnormal increase in superficial SA colonization may be one of the reasons for the increase in CD4+ Trm cell number at SLE skin lesions.In this study,we investigated the effect of abnormally increased superficial skin SA on T and B cell subsets of d LN and spleen,humoral immunity and local CD4+ Trm cells in a NP-KLH model and an IMQinduced mouse model of lupus-like disease.To further elucidate whether the abnormally increased SA in lupus lesions has any effect on T and B cell subsets,autoantibody production and local CD4+ Trm cell composition in the skin through animal models.Part Ⅰ The impact of superficial Staphylococcus aureus skin colonization on NP-KLH-induced humoral immune responseObjective:To investigate the effect of superficial SA skin colonization on NPKLH-induced humoral immune response and the composition of CD4+Trm cells in the skin at the colonization site.Methods:1.To explore the effects of SA superficial skin colonization on a continuous NP-KLH immune induction model:(1)To explore whether superficial colonization of SA affects the humoral immune response to NP-KLH under continuous immunization,we immunized mouse with 100 μL(1mg/m L)of NPKLH subcutaneously in the buttocks on day 0 and day 7,and then immunized them with the same dose of NP-KLH at the same site on days 21,35,and 41.We then performed epidermal stripping and superficial colonization of SA/PBS on two groups of mouse during days 8-15 in the early stage of modeling and days 42-51 in the late stage of modeling.(2)ELISA was used to detect mouse serum Anti-NP levels at different time points.(3)Flow cytometry was used to analyze T and B cell subsets in lymph nodes and spleen.2.Clarify the time window in which serum Anti-NP antibodies levels change after superficial SA intervention:(1)Because the SA group showed a slightly lower level of anti-NP antibody in a short time after the first SA implantation in the continuous NP-KLH immunization.To further confirm the effect of SA,we conducted back interventions of SA/PBS on the day 3 after NP-KLH immunization on day 0 and day 7.Mouse were sacrificed on the Day 3,Day 8 and Day 21 after intervention.(2)ELISA was used to detect mouse serum levels of Anti-NP subclasses and total Ig G,Ig M,Ig G1,Ig G2 a and Ig G2 b at different time points.(3)ELISA was used to detect the affinity of mouse serum Anti-NP Ig G at different time points.(4)Flow cytometry was used to analyze T and B cell subgroups in lymph nodes and spleen.(5)Flow cytometry was used to analyze the CD4+ cell subpopulation in the skin at the intervention site on day 21 after the SA/PBS intervention.Results:1.The effect of SA superficial skin colonization on a continuous NPKLH immune induction model:(1)In continuous NP-KLH immunization induction,we observed that the serum Anti-NP Ig M,Ig G and Ig G2 b levels in the SA group were slightly lower than those in the PBS group in a short time after the end of SA superficial implantation on the back,but the difference was not statistically significant(PBS: n = 5,SA: n = 5,P > 0.05).(2)Under continuous NP-KLH immunization,compared with PBS group,SA group had significantly increased number of CD19+ B,Na(?)ve B,Memory B,GC B,Tfh,and Th17 in d LN and spleen(P <0.05).SA group had significantly increased Plasmablast and Plasma cell in the d LN and decreased Plasmablast and Plasma cell in spleen(PBS: n = 5,SA: n = 5,P < 0.05).2.Time window in which serum Anti-NP antibodies levels change after superficial SA intervention:(1)On the 8th day after SA/PBS intervention,Anti-NP Ig G2 a and Ig G2 b in the SA group were significantly lower than those in the PBS group(PBS: n = 5,SA: n = 7,P < 0.05),Anti-NP Ig M,Ig G1,and Ig G were also slightly lower than those in the PBS group(PBS:n = 5,SA: n = 7,P > 0.05);further detection of serum total Ig M,Ig G,Ig G1,Ig G2 a,and Ig G2 b found that on the 8th day when the serum levels of each subclass of Anti-NP specific antibodies were significantly decreased,there was no significant difference in the total antibody subclasses between the two groups,furthermore,SA group had an increase in serum Ig G1(PBS: n = 5,SA: n = 7,P >0.05).(2)Compared with PBS control,the affinity of serum Anti-NP Ig G generated by SA group was significantly higher than that on the 3rd and 8th day(PBS: n = 5,SA: n = 7,P < 0.01).(3)After SA/PBS intervention,B cell subpopulations of SA group significantly increased in the d LN on the 3rd,8th,and 21 st days(PBS: n = 5,SA: n = 7,P < 0.05),while there was no significant difference in the spleen between two group(PBS: n = 5,SA: n = 7,P > 0.05).(4)After SA/PBS intervention on the 3rd day,the number of Th17 cells of SA group significantly increased in the d LN and spleen;on the8 th day,the number of Th1,Th2,and Th17 cells of SA group significantly increased in the DLN and spleen;on the 21 st day,the number of Th1,Th2,and Th17 cells of SA group significantly increased in the d LN,and only Th17 cells of SA group significantly increased in the spleen(PBS: n = 5,SA: n = 7,P < 0.05).(5)On the 21 st day after SA/PBS intervention,the number of CD4+ and CD4+ Trm cells in the skin of SA group increased significantly.The number of Th subgroups,especially Th17,in the skin of SA group increased significantly(PBS: n = 5,SA: n = 7,P < 0.05).Summary:SA superficial colonization on the back can induce B cell proliferation and differentiation in lymph nodes while specifically decreasing the serum level of various subclasses of Anti-NP antibodies in a short period of time.After SA superficial implantation on the back,the number of CD4+ and CD4+ Trm cells,especially Th17,in the local skin were significantly increased.Part Ⅱ The impact of superficial Staphylococcus aureus skin colonization on the development of lupus-like model induced by IMQ in earObjective:To investigate the effect of superficial SA skin colonization on the development of lupus-like model induced by IMQ in ear and the composition of CD4+ Trm cells in the colonized skin.Methods:1.Phenotypic analysis of the IMQ-induced lupus model affected by superficial SA skin colonization:(1)Lupus-like model was induced by IMQ in ear,and SA/PBS superficial planting was performed on the back of lupus mouse for1 week before and after IMQ was performed,1 week before the end of modeling.(2)The progress of kidney damage was evaluated by monitoring urinary protein.(3)ELISA was used to detect the serum Anti-ds DNA Ig G levels of mice at different time points.2.Mouse kidney pathology assessment:(1)H&E staining for pathological changes in the kidney at the modelling endpoint.(2)Immunofluorescence staining for C3 and Ig G deposits in the kidney.3.Flow cytometry was used to analyze T and B cell subsets in d LN and spleen.4.Flow cytometry was used to analyze the CD4+ cell subpopulation in the skin at the SA/PBS intervention site.Results:1.Phenotypic analysis of the IMQ-induced lupus model affected by superficial SA skin colonization:(1)Superficial colonization of SA on the back leads to a significant increase of SA without causing irreversible damage to the skin(Blank: n = 5,PBS: n = 5,SA: n = 15,P < 0.05).(2)In the 4th-5th week of IMQ administration,the urinary protein level of mouse in the PBS group was significantly higher than that in the SA group(Blank: n = 5,PBS: n = 5,SA: n = 15,P < 0.05).(3)In the later stage of modeling,the serum Anti-ds DNA Ig G level of mice in the SA group was slightly lower than that in the PBS group(Blank: n = 5,PBS: n = 5,SA: n = 15,P > 0.05).2.Mouse kidney pathology assessment:(1)H&E staining of kidney tissue of 3 groups of mice showed that compared with the Blank group,the glomeruli of mice in the PBS group and SA group were significantly enlarged,but there was no significant difference in glomeruli between the PBS group and SA group.(2)C3 and Ig G immunofluorescence staining of kidney showed that there was no C3 and Ig G immune complex deposition in the glomeruli of mice in the Blank group,while the kidneys of the PBS and SA groups had similar degrees of C3 and Ig G immune complex deposition(Blank: n = 5,PBS: n = 5,SA: n = 15,P > 0.05).3.T and B cell subsets in lymph nodes,spleen: There was no significant difference in the subgroups of T and B cells in the d LN and spleen of mouse between SA group and PBS group(Blank: n = 5,PBS: n ≥ 5,SA: n ≥ 15,P > 0.05).4.CD4+ cell subpopulation in skin at SA/PBS intervention sites:(1)Compared with the blank group,the number of CD4+,Th1,Th17,Treg,CD4+ Trm,Th1-like Trm,Th2-like Trm and Th17-like Trm in the SA group was significantly increased(Blank: n = 5,PBS: n = 7,SA: n = 15,P < 0.05).(2)Compared with the blank group,there was no significant change in the number of CD4+,Th1,Th17,Treg,CD4+ Trm,Th1-like Trm,Th2-like Trm and Th17-like Trm in the PBS group(Blank: n = 5,PBS: n = 7,SA: n = 15,P < 0.05).(3)Compared with the PBS group,the number of CD4+,Th1,Th2,Th17 and Treg in the SA group was significantly increased,and the skin barrier is dominated by Th17 cells;the number of subgroup of CD4+ Trm was significantly increased,and especially Th17-like Trm and Treg-like Trm(Blank: n = 5,PBS: n = 7,SA: n = 15,P <0.05).Summary:SA induced an increased CD4+ cells in skin characterized by large proportion of Th17 and Th17-like Trm cells,and the maintenance was not relied on persistence of SA.In contrast,the effects of SA on urinary protein,serum Anti-ds DNA Ig G levels,lymph node and spleen immune cells in the mouse lupus-like model faded as the ability of SA to colonize the skin diminished.Part Ⅲ The impact of superficial Staphylococcus aureus skin colonization on the development of lupus-like model induced by IMQ at the same siteObjective:To investigate the effect of superficial SA skin colonization on the development of lupus-like model induced by IMQ at the same site and the composition of CD4+ Trm cells in the colonized skin.Methods:1.To explore the effect of recent(2 weeks before modelling)superficial SA skin colonization on IMQ-induced lupus models:(1)In the 8-week model,to explore the short-term effect of SA superficial skin colonization on the model,we used IMQ to induce lupus-like model by back administration.Two weeks before IMQ intervention,SA/PBS was planted superficially at the IMQ administration site for 1 week,and SA/PBS was planted superficially 1 week again before the end of modeling.(2)The progress of kidney damage was evaluated by monitoring urinary protein.(3)ELISA was used to detect the serum Anti-ds DNA Ig G,Anti-ANA Ig G levels of mice at different time points.(4)Mouse kidney pathology assessment: H&E staining for pathological changes in the kidney at the modelling endpoint.Immunofluorescence staining for C3 and Ig G deposits in the kidney.(5)Flow cytometry was used to analyze T cell infiltration in the kidney.(6)Flow cytometry was used to analyze T and B cell subsets in d LN and spleen.(7)Flow cytometry was used to analyze the CD4+ cell subpopulation in the skin at the SA/PBS intervention site.2.To explore the effect of previous(4 weeks before modelling)superficial SA skin colonization on IMQ-induced lupus models:(1)In the 11-week model,to explore the long-term effect of SA superficial skin colonization on the model,we used IMQ to induce lupus-like model by back administration.4 weeks before administration and 1 week before the end of modeling,SA/PBS was planted superficially at the IMQ administration site for 1 week.(2)The progress of kidney damage was evaluated by monitoring urinary protein.(3)ELISA was used to detect the levels of serum Anti-ds DNA Ig G,total Ig G,Ig G1,Ig G2 a,Ig G2 b and Ig M in mice at different time points.(4)Flow cytometry was used to analyze the CD4+ cell subpopulation in the skin at the SA/PBS intervention site.Results:1.The effect of recent(2 weeks before modelling)superficial SA skin colonization on IMQ-induced lupus models:(1)Urinary protein levels in the PBS group were significantly higher than those in the SA group at the 6th week of IMQ administration(PBS: n = 6,SA: n = 6,P < 0.05).(2)Serum Anti-ds DNA Ig G in the SA group was selectively decreased during the 4th-6th week of IMQ administration(PBS: n = 6,SA: n= 6,P < 0.01).Starting from the 2nd week of IMQ administration,serum Anti-ANA Ig G was slightly lower in the SA group than in the PBS group(PBS: n = 6,SA: n = 6,P > 0.05).(3)H&E staining showed no significant difference in glomeruli between PBS and SA.C3 and Ig G immunofluorescence staining of kidney showed no significant difference in C3 and Ig G immune complex deposition in the glomeruli of mice in the PBS group and SA group.(4)The number of CD4+ cells infiltrating the kidney in the PBS group was significantly higher than that in the SA group(PBS: n = 6,SA:n = 6,P < 0.0001).(5)The number of CD19+ B cells in the spleen of SA group were decreased,the proportion of Memory B cells in the spleen of SA group were increased,and the number of Plasmablast and Plasma cells in the spleen of SA group were increased;the number of Th17 cells in the d LN of SA group were increased(PBS: n = 6,SA: n = 6,P < 0.05).(6)Scaling appeared at the IMQ administration site of mouse in the SA group 2 days after the first IMQ administration.At the end of modeling,the proportion of infiltrating CD4+ cells and CD4+ Trm cells in the skin of SA group were increased significantly(PBS: n =6,SA: n = 6,P < 0.05).2.The effect of previous(4 weeks before modelling)superficial SA skin colonization on IMQ-induced lupus models:(1)During the 5th,7th and 9th weeks of IMQ administration,urinary protein levels in the PBS group were slightly higher than those in the SA group(PBS: n = 9,SA: n = 11,P > 0.05).(2)During the 9th and 10 th weeks of IMQ administration,serum Antids DNA Ig G in the SA group was selectively decreased,but the effect was not as good as that in the 8-week model(PBS: n = 9,SA:n = 11,P > 0.05).(3)Compared with PBS group,the number of infiltrating CD3+CD4+and CD3+CD4-cells in the skin of SA group were significantly increased,and there was no significant difference in the proportion of CD4+ Trm cells.Among CD4+ subgroups,Th1 and Th17 cells in the SA group were significantly increased,while the proportion of Treg cells were decreased significantly.Among CD4+ Trm subgroups,Th1-like Trm,Th2-like Trm and Th17-like Trm cells in SA group were significantly increased,while the proportion of Treg-like Trm cells decreased significantly(PBS: n = 8,SA: n = 11,P < 0.05).Summary:Superficial colonization of SA selectively reduced Anti-ds DNA Ig G serum levels in IMQ-induced lupus mouse model,suppressed urinary protein and significantly reduced renal CD4+ cell infiltration,but had no significant effect on glomerular lesions.IMQ stimulation of SA colonization sites further induced an increase in the proportion of Th1,Th1-like Trm,and Th2-like Trm cells in the skin.Conclusion:1.SA superficial colonization on the back can induce B cell proliferation and differentiation in lymph nodes while specifically decreasing the serum level of various subclasses of Anti-NP antibodies in a short period of time.2.Superficial colonization of SA selectively reduced Anti-ds DNA Ig G serum levels in IMQ-induced lupus mouse model,suppressed urinary protein and significantly reduced renal CD4+ cell infiltration,but had no significant effect on glomerular lesions.3.SA induced an increased CD4+ cells in skin characterized by large proportion of Th17 and Th17-like Trm cells,and the maintenance was not relied on persistence of SA.IMQ stimulation of SA colonization sites further induced an increase in the proportion of Th1,Th1-like Trm,and Th2-like Trm cells in the skin. |
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