endocervical cell line  (ATCC)

Journal: Scientific Reports

Article Title: Modeling of variables in cellular infection reveals CXCL10 levels are regulated by human genetic variation and the Chlamydia- encoded CPAF protease

doi: 10.1038/s41598-020-75129-y

Figure Lengend Snippet: Suppression of CXCL10 following C. trachomatis infection is mediated by the pathogen-encoded protease, CPAF. ( a , b ) Chemically generated CPAF mutant increases levels of CXCL10 compared to WT C. trachomatis infection. A2ENs ( a ) and three LCLs ( b ), HG01507, HG01519, and HG01527 were infected with a CPAF + (RST5) or CPAF − (RST17) LGV-L2 strain of C. trachomatis at MOI 5. At 70 h post infection, cell culture supernatants were harvested and assayed for CXCL10 concentration by ELISA. Data are from 12 biological replicates per condition across 2 experiments. P-values are from unpaired t-tests. ( c ) DNA gel confirming ~ 1.5 kb TargeTron-mediated insertion in cpa :: cat LGV-L2 strain. ( d ) TargeTron-generated CPAF mutant increases fold change of CXCL10 during C. trachomatis infection. 23 LCLs were infected at MOI 5 with cpa :: cat LGV-L2 or WT parental LGV-L2. Cell culture supernatants were collected at 70 h post infection and assayed by ELISA. Data is represented as log 2 ((infected [CXCL10] pg/ml)/(uninfected [CXCL10] pg/ml)). Each point is the mean from 3 biological replicates of each LCL across 3 experiments. P-values are from paired t-test. ( e ) Dose–response of infection with cpa :: cat LGV-L2 or WT parental LGV-L2 in HG01519 and HG02647. Cell culture supernatants were collected at 72 h post infection and assayed by ELISA. Increasing MOI from 2 to 15 results in greater CXCL10 suppression in the WT strains while cpa :: cat LGV-L2 strain demonstrates increasing CXCL10 with increasing MOI. P-values are from two-way ANOVA comparing cpa :: cat LGV-L2 and WT LGV-L2 infected values. Note that for HG02647, the effect of CPAF is apparent at all MOIs resulting in a significant p-value for the effect of strain (p < 0.0001). For HG01519, the effect of CPAF is only apparent at the highest MOI, resulting in a significant p-value (p = 0.0039) for the interaction of strain and MOI. ( f , g ) Time-course of infection with cpa :: cat LGV-L2 or WT parental LGV-L2 at MOI 5 in HG01519 and HG02647 ( f ) or A2EN ( g ). Cell culture supernatants were collected at 24, 48, 72, and 96 h post infection and assayed by ELISA. Similar levels of CXCL10 were detected at 24 and 48 h and increasing levels of CPAF-dependent suppression is observed from 72 to 96 h. P-values are from two-way ANOVA comparing cpa :: cat LGV-L2 and WT LGV-L2 infected values. Data for E, F and G are from 4 biological replicates per condition from 2 experiments.

Article Snippet: Human Endocervical Epithelial Cell Line, A2ENs obtained from Raphael Valdivia, were grown in defined KSFM media (Thermo; 10744019) supplemented with 10% FBS.

Techniques: Infection, Generated, Mutagenesis, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Modeling of variables in cellular infection reveals CXCL10 levels are regulated by human genetic variation and the Chlamydia- encoded CPAF protease

doi: 10.1038/s41598-020-75129-y

Figure Lengend Snippet: Modeling fold change of cytokine concentration as a function of bacterial burden reveals CPAF-independent suppression of RANTES. ( a ) All measured cytokines except CXCL10 demonstrate an average increase in concentration following C. trachomatis infection. 70 h-post-infection cell culture supernatants from 528 LCLs assayed by Luminex for 17 cytokines (log 2 (infected [cytokine]/uninfected [cytokine]). ( b ) CXCL10 and RANTES demonstrate a negative correlation with bacterial burden. Pearson correlation between 46 h-post-infection % infected cells and log2(infected [cytokine]/uninfected [cytokine]) for 17 cytokines, correlation is calculated over 528 LCLs. ( c ) Scatter plot of fold change of RANTES (log 2 ((Infected [RANTES] pg/ml)/(Uninfected [RANTES] pg/ml))) vs. 46hpi % infected cells showing a highly significant negative correlation (Pearson correlation p < 0.0001; r = -0.27). ( d ) Linear modeling of fold change of RANTES (log2((Infected [RANTES] pg/ml)/(Uninfected [RANTES] pg/ml))) as a function of population, 70hpi cell death, and 46hpi %infected cells showing that C. trachomatis burden has a larger effect on fold change of RANTES than population or cell death. 528 lymphoblastoid cell lines from 4 populations in parent–offspring trios were infected with C. trachomatis LGV-L2 RifR pGFP::SW2. At 70 h post infection, cell culture supernatants were harvested and RANTES protein quantified by Luminex. ( e ) Distribution of suppression and induction of RANTES after C. trachomatis infection show most LCLs have an overall increase in RANTES while 94 LCLs demonstrate a decrease. ( f ) CPAF does not regulate concentration of RANTES. Three LCLs, HG01507, HG01519, and HG01527 and A2ENs were infected with CPAF + (RST5) or CPAF- (RST17) LGV-L2 strain of C. trachomatis . At 70 h post infection cell culture supernatants were harvested and assayed for RANTES concentration by ELISA. Data are from 12 biological replicates across 2 experiments. P-values are from unpaired t-tests.

Article Snippet: Human Endocervical Epithelial Cell Line, A2ENs obtained from Raphael Valdivia, were grown in defined KSFM media (Thermo; 10744019) supplemented with 10% FBS.

Techniques: Concentration Assay, Infection, Cell Culture, Luminex, Enzyme-linked Immunosorbent Assay