Fig 1.
T cell phenotyping and parasite load in WT B6 and CD3e−/− mouse following S. japonicum infection.
(A) Representative FACS gating of immune cells in spleen from wild-type (WT) B6 and CD3e−/− mice. Expression of CD4 and CD8 by T cells (CD45+CD3+TCRβ+; identified at left) from the blood of WT B6 (top) and CD3e−/− mice (bottom). Numbers adjacent to outlined areas indicate percentage of cells in each gate. (B, C) Total numbers of T cells of blood (B) or spleen samples (C) from CD3e−/− mice compared with those from WT B6 mice uninfected (top) or 6 weeks post-infection (bottom). Data of infected mice are from two independent experiments (uninfected condition: WT, n = 3; CD3e−/−, n = 6, infected: WT, n = 10; CD3e−/−, n = 9). (D) Comparison of liver pathological changes in B6 (left) and CD3e−/− mouse (right) at 6 weeks post-infection. (E) Representative micrographs of worms harvested from portal and mesenteric veins of infected B6 mouse (top) and CD3e−/− mice (bottom) 6 weeks after cercariae infection (Scale bar, 5 mm.). (F) Width of worms collected from portal and mesenteric veins of WT and CD3e−/− mouse 6-week after infection was determined from digital micrographs. Data are from three experiments (WT, n = 34; CD3e−/−, n = 19). (G) Length of worms recovered from WT and CD3e−/− mice at 6-week post-challenge was measured based on digital micrographs (WT, n = 15; CD3e−/−, n = 20). (H) Number of worms recovered from WT and CD3e−/− mice at 6 weeks after challenge (WT, n = 11; CD3e−/−, n = 11). (I) The recovery rate of S. japonicum collected from portal and mesenteric veins of infected B6 mouse and CD3e−/− mouse. Data were from three independent experiments (WT, n = 11; CD3e−/−, n = 11). The following fluorochrome-tagged antibodies were used for FACS analyses: anti-mouse CD45-APC-eFluor 780, anti-mouse CD4-PE-Cyanine7, anti-mouse CD3ε-PE-Cyanine5.5, anti-mouse TCR beta-FITC, anti-mouse CD8α -Alexa Fluor 700. Data represent the mean ± s.e.m. Statistical significance was assessed by unpaired Student’s t-test or non-parametric unpaired Mann-Whitney test and indicated by * P<0.05, **** P<0.0001, NS, non-significant.
Fig 2.
Immunopathologic characteristics of SD rat after S. japonicum infection.
(A) S. japonicum infection does not cause overt liver granuloma in SD rat, liver of uninfected SD rat (left) and liver of infected (right). (B) Representative micrographs of worms collected from portal and mesenteric veins of B6 mouse (left) and SD rat (right) both infected with S. japonicum for 6 weeks (Scale bar, 5 mm). (C) Width of worms from B6 mice and SD rats at week 6 post-challenge was measured based on digital micrographs. Data are from two experiments (B6 mouse, n = 5; SD rat, n = 6). (D) Length of worms harvested from portal and mesenteric veins of infected B6 mouse and SD rat was determined. Data are from two independent experiments (B6 mouse, n = 15; SD rat, n = 33). (E) Number of male and female worms recovered from B6 mouse and SD rat at 6-week post-challenge. (F) Percentage of worm recovery from portal and mesenteric veins of infected B6 mouse and SD rat, presented from two experiments (B6 mouse, n = 7; SD rat, n = 6). (G-I) Representative FACS plots of immune cells in spleen (G) or peripheral blood (I) of SD rat under uninfected and infected conditions. (H, J) Absolute number of total T cells, CD4+ or CD8+ T cells in G and I. Data are from two independent experiments (H, uninfected, n = 7; infected, n = 8. J, uninfected, n = 7; infected, n = 10). The following fluorochrome-tagged antibodies were used: anti-rat CD45 -eFluor450, anti-rat CD3-APC, anti-rat CD4-APC/Cyanine7, anti-rat CD8α-PE. Data represent the mean ± s.e.m. Statistical significance was assessed by unpaired Student’s t-test or non-parametric unpaired Mann-Whitney test and indicated by * P<0.05, ** P<0.01, **** P<0.0001, NS, non-significant.
Fig 3.
Generation and analysis of Lck knock-out SD rat.
(A) Schematic diagram of the rat Lck locus and sgRNA targeting sequences. sgRNA 1 sequence located in exon 3 and sgRNA 2 in exon 4 were indicated respectively. (B) DNA sequencing analysis of mutant alleles from two Lck mutant founders (KO#1 and KO#2) confirms genome editing. Sanger sequencing results showed large-fragment deletions indicated in red dashes. Sequences non shown between sgRNA 1 and sgRNA 2 were as indicated in black dots. The protospacer adjacent motifs (PAM) were indicated in red letters in A or B. (C) Expression of CD4 and CD8 T cells by WT rat and Lck−/− rat thymocytes (left), analyzed by flow cytometry. Absolute number of white blood cell (WBC), CD4+ or CD8+ single positive (SP) cells, double-negative (DN) CD4−CD8− thymocytes and double-positive (DP) CD4+CD8+ thymocytes (right). (D, E, F) Representative flow cytometry of lymphocytes in peripheral blood (D), spleen (E), or lymph nodes (F) of WT SD rat and Lck−/− SD rat (left). Numbers adjacent to outlined areas (left) indicate percent cells in each gate; absolute number of WBC, CD3+, CD4+ or CD8+ T cells in blood (D), spleen (E), or lymph nodes (F) of WT SD rat and Lck−/− SD rat (right). C−F, numbers adjacent to outlined areas indicate percentage of each population (left); data from two independent experiments (n = 5 per group). The following fluorochrome-tagged antibodies were used: anti-rat CD45-eFluor450, anti-rat CD3-APC, anti-rat CD4-APC/Cyanine7, anti-rat CD8α-PE. Data represent the mean ± s.e.m. Statistical significance was assessed by unpaired Student’s t-test or non-parametric unpaired Mann-Whitney test and indicated by ** P<0.01, *** P<0.001, **** P<0.0001, NS, non-significant.
Fig 4.
Immunopathologic analysis of Lck−/− SD rat following schistosome infection.
(A) S. japonicum skin infection does not cause noticeable liver granuloma in Lck−/− SD rat (right) compared to the liver of infected WT rat control (left). (B) H&E staining of liver sections from uninfected or infected WT SD rat and Lck−/− SD rat. (Original magnification, ×100; Scale bar, 100 μm). (C, D) Granuloma size was determined based on H&E staining of liver sections using CaseViewer software (WT rat, n = 6; Lck−/− rat, n = 5). (E) Representative micrographs of S. japonicum recovered from portal and mesenteric veins of infected WT SD rat (left) or Lck−/− SD rat (right) at 42 days after infection (Scale bar, 5 mm). (F) Width of worms from WT rat and Lck−/− rat at week 6 post-challenge was measured based on digital micrographs (worms from WT rats, n=8; worms from Lck−/− rat, n = 8). (D) Length of worms harvested from infected WT SD rat and Lck−/− SD rat. Data are from two independent experiments (worms from WT rat, n = 25; worms from Lck−/− rat, n = 15). (H) Number of male and female worms recovered from WT and Lck−/− SD rat at 6-week post-infection. (I) The recovery rate of S. japonicum collected from infected WT rat and Lck−/− SD rat. Data are from two independent experiments (WT rat, n = 6; Lck−/− SD rat, n = 13). Data represent the mean ± s.e.m. Statistical significance was assessed by unpaired Student’s t-test or non-parametric unpaired Mann-Whitney test and indicated by * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.
Fig 5.
Pathologic analysis of WT and Lck−/− rat after S. japonicum infection.
(A) Masson’s trichrome staining of liver sections from uninfected or infected WT and Lck−/− rat. (Original magnification, ×100; Scale bar, 100 μm). (B) Fibrotic areas in Masson’s trichrome staining of liver sections using CaseViewer software (WT, n = 6; Lck−/−, n = 5). (C) qPCR analysis of mRNA expression for fibrosis-related genes in liver of uninfected or infected WT and Lck−/− rats: Col1α1, Col3α1 and α-SMA. (D) CD11b staining of liver sections from uninfected or infected WT and Lck−/− rat (left); CD11b+ area shown as percentage measured from CD11b stained liver sections using CaseViewer software (right) (WT, n = 5; Lck−/−, n = 5). Original magnification, ×100; Scale bar, 100 μm. (E) CD19 staining of liver sections from uninfected or infected WT and Lck−/− rat (left); CD19+ area in percentage measured by CD19 staining of liver sections using CaseViewer software (right) (WT, n = 5; Lck−/−, n = 5). Original magnification, ×100; Scale bar, 100 μm. Statistical significance was assessed by unpaired Student’s t-test or non-parametric unpaired Mann-Whitney test and indicated by * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.
Fig 6.
Comparison of Treg frequency and transcription factors expression of B6 mouse and SD rat upon S. japonicum infection.
(A, C) FACS analysis of FOXP3+ Tregs from B6 mouse (A) or SD rat (C) under uninfected and infected conditions. Expression of FOXP3 on CD4+ T cells from uninfected and 6-week S. japonicum-infected mice or rat. (B) Quantification of data in A. Data is representative of two experiments (uninfected B6 mouse, n = 8; infected B6 mouse, n = 10). (D) Quantification of data in C. Results represent for three experiments (uninfected rat, n = 8; infected rat, n = 14). (E, F) mRNA expression of transcription factor TBX21 (T-bet), GATA3, RORC or BCL6 of FACS-sorted CD4+ T cells from B6 mouse (E) or SD rat (F) under uninfected and infected conditions by using real-time PCR. Hprt or Sdha was used as internal controls for mice or rat, respectively. Relative mRNA levels are presented as mean ± s.e.m. The following fluorochrome-tagged antibodies were used: anti-mouse CD45-APC-eFluor 780, anti-mouse CD5-eFluor 450, anti-mouse CD4-Brilliant Violet 605, anti-mouse FOXP3-PE, anti-rat CD45-eFluor450, anti-rat CD3-APC, anti-rat CD4-APC/Cyanine7, anti-rat FOXP3-PE. Data represent the mean ± s.e.m. Statistical significance was assessed by unpaired Student’s t-test or non-parametric unpaired Mann-Whitney test and indicated by *, P<0.05; **, P<0.01; ***, P<0.0001; NS, non-significant.