TRIF mediates TLR2 proinflammatory signaling. (A) BMDM isolated from wild-type (WT) and TLR2-deficient mice (TLR2^−/−) were stimulated with 1 μg of Pam₃CSK₄/ml or B. burgdorferi at MOI of 10, in the presence of polymyxin B for 6 h. Expression of ifn-β, tnf-α, cxcl10, and il6 was measured by RT-qPCR. Values represent mean induction of expression relative to control cells. (B) BMDM isolated from wild-type (WT) and TRIF-deficient mice (TRIF^−/−) were stimulated with 1 μg of Pam₃CSK₄/ml, 10 μg of poly(I·C)/ml and 5 ng of LPS/ml for 6 h. Except for LPS, stimulations were performed in the presence of polymyxin B. Values represent mean secretion of IL-6 in deficient BMDM relative to wild-type cells measured by ELISA. For IL-6, wild-type cells stimulated with Pam₃CSK₄ secreted a mean of 2.52 ng/ml, TRIF-deficient cells secreted a mean of 0.47 ng/ml. Wild-type cells stimulated with poly(I·C) secreted a mean of 1.3 ng of IL-6/ml, whereas TRIF-deficient cells secreted no detectable (ND) levels. Wild-type cells stimulated with LPS secreted a mean of 7.08 ng of IL-6/ml, while TRIF-deficient cells secreted a mean of 0.56 ng/ml. *, P < 0.05 by Mann-Whitney U. (C) BMDM isolated from wild-type (WT), MyD88-deficient mice (MyD88), TRIF-deficient mice (TRIF), and MyD88 and TRIF doubly deficient mice (DKO) were stimulated with Pam₃CSK₄ at 1 μg/ml. For IL-6, measured by ELISA at 6 h postinfection, wild-type cells secreted a mean of 2.52 ng/ml, MyD88-deficient cells had undetectable levels (ND), TRIF-deficient cells secreted a mean of 0.47 ng/ml and DKO cells had undetectable levels. *, P < 0.05 by Mann-Whitney U. For TNF-α, measured by ELISA at 6 h postinfection, wild-type cells secreted a mean of 1.87 ng/ml, MyD88-deficient cells and DKO-deficient cells had no detectable levels, and TRIF-deficient cells secreted a mean of 0.8 ng/ml. *, P < 0.05 by Mann-Whitney U. For IL-10, measured by ELISA at 16 h postinfection, wild-type cells secreted a mean of 0.99 ng/ml, MyD88-deficient cells, and DKO-deficient cells had no detectable levels and TRIF-deficient cells secreted a mean of 0.61 ng/ml. *, P < 0.05 by Mann-Whitney U. (D) BMDM isolated from wild-type (WT), MyD88-deficient mice (MyD88), TRIF-deficient mice (TRIF) and MyD88 and TRIF doubly deficient mice (DKO) were stimulated with B. burgdorferi at an MOI of 10. For IL-6, measured by ELISA at 6 h postinfection, wild-type cells secreted a mean of 2.64 ng/ml, MyD88- and DKO-deficient cells had no detectable (ND) levels, TRIF-deficient cells secreted a mean of 0.99 ng/ml. *, P < 0.05 by Mann-Whitney U. For TNF-α, measured by ELISA at 6 h postinfection, wild-type cells secreted a mean of 1.81 ng/ml, MyD88-deficient cells and DKO-deficient cells had no detectable levels and TRIF-deficient cells secreted a mean of 0.98 ng/ml. *, P < 0.05 by Mann-Whitney U. For IL-10, measured by ELISA at 16 h postinfection, wild-type cells secreted a mean of 1.28 ng/ml, MyD88-deficient cells a mean of 0.39 ng/ml, DKO-deficient cells a mean of 0.19 ng/ml, and TRIF-deficient cells secreted a mean of 0.3 ng/ml. *, P < 0.05 by Mann-Whitney U.

TRIF mediates activation of type I interferons in response to TLR2 ligands. (A) BMDM isolated from wild type (WT), MyD88-deficient mice (MyD88), TRIF-deficient mice (TRIF), and MyD88 and TRIF doubly deficient mice (DKO) were stimulated with 1 μg of Pam₃CSK₄/ml. Expression of ifn-β, cxcl10 and ifit1, measured at 6 h postinfection, and ifn-α, measured at 16 h postinfection, was determined by RT-qPCR. Values represent mean induction of expression relative to control cells and the standard errors of the mean (SEM) of three independent experiments. *, P < 0.05 by Mann-Whitney U. (B) BMDM isolated from wild type (WT), MyD88-deficient mice (MyD88), TRIF-deficient mice (TRIF), and MyD88 and TRIF doubly deficient mice (DKO) were stimulated with B. burgdorferi at an MOI of 10. Expression of ifn-β, ifn-α, cxcl10, and ifit1 was measured by RT-qPCR. Expression of ifn-β, cxcl10, and ifit1, measured at 6 h postinfection, and ifn-α, measured at 16 h postinfection, was determined by RT-qPCR. Values represent mean induction of expression relative to control cells and SEM of three independent experiments. *, P < 0.05 by Mann-Whitney U. (C) BMDM isolated from wild-type (WT) and TRIF-deficient (TRIF^−/−) mice were stimulated with 5 ng of LPS/ml. Expression of ifn-β, cxcl10, and ifit1, measured at 6 h postinfection, and ifn-α, measured at 16 h postinfection, was determined by RT-qPCR. Values represent mean induction of expression relative to control cells and the SEM of three independent experiments. *, P < 0.05 by Mann-Whitney U. (D) BMDM isolated from wild-type (WT) and TRIF-deficient (TRIF) mice were stimulated with 1 μg of Pam₃CSK₄/ml and B. burgdorferi at an MOI of 10 for 6 h. Expression of irf7 was measured by RT-qPCR. Values represent the mean induction of expression relative to control cell and the SEM of three independent experiments. *, P < 0.05 by Mann-Whitney U.

TRIF deficiency has no effect on the control of B. burgdorferi infection in vivo or B. burgdorferi phagocytosis in vitro. (A) WT (n = 10), MyD88-deficient (n = 10), TRIF-deficient (n = 10), and MyD88/TRIF doubly deficient (DKO) mice (n = 8) were infected with 10⁵ bacteria and sacrificed at 3 weeks postinfection. Bacterial loads in joints were quantified by qPCR for the bacterial flaB gene and normalized to copies of mouse β-actin. For MyD88 and DKO mice, P < 0.0001 by Mann-Whitney U. (B) BMDM from wild-type and TRIF-deficient mice were stimulated with B. burgdorferi at an MOI of 10 for 60 min and stained by immunofluorescence as described in panel C. Percent internalized B. burgdorferi was determined by counting numbers of cells with internalized bacteria versus cells not containing internalized bacteria. The data represent the mean of internalized bacteria ± the SEM of three independent experiments. *, P < 0.05 by Mann-Whitney U. (C) Internalization of spirochetes in wild-type (WT) or TRIF-deficient cells was determined by immunofluorescent staining before (A488/green-labeled) and after (A549/red-labeled) permeabilization of the cells. Nuclei are stained with DAPI.

MyD88, but not TRIF deficiency, has an effect on expression of MARCO in vitro and in vivo and B. burgdorferi phagocytosis is impaired in MARCO-deficient mice. (A) Wild-type and MyD88-deficient BMDM were incubated with B. burgdorferi at an MOI of 10 for 5, 20, and 60 min. Cells were harvested and processed for RNA using TRIzol. RT-qPCR was performed using primers for Lox-1, SR-A, and MARCO and values represent mean induction of expression relative to wild-type unstimulated cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Mann-Whitney U. (B) BMDM from wild-type and MARCO-deficient mice were stimulated with B. burgdorferi at an MOI of 10 for 60 min. Internalization of spirochetes was determined by immunofluorescent staining before (blue) and after (red) permeabilization of the cells. Lysosomes were stained with anti-Lamp1 antibodies (green). (C) Percent internalized B. burgdorferi was determined by counting numbers of cells with internalized bacteria versus cells not containing internalized bacteria. The data represent the means of internalized bacteria ± the SEM of three independent experiments. *, P < 0.05 by Mann-Whitney U. (D) Expression of MARCO in wild-type, TRIF-deficient, and MyD88-deficient BMDM stimulated with B. burgdorferi at an MOI of 10 was measured by RT-qPCR. Values represent mean induction of expression relative to control cells and the SEM of three independent experiments. *, P < 0.05 by Mann-Whitney U. (E) Levels of marco transcript were measured by RT-qPCR from joint tissue in wild type (n = 10), TRIF-deficient (n = 10), MyD88-deficient (n = 10), and doubly deficient (n = 9) mice. One mouse in the wild-type group was arbitrarily set to a value of 1, and all other values are shown in reference to that value using the 2^−ΔΔCT method after normalization for the value of β-actin. Each circle in the data represents one mouse, and the lines indicate median values. P < 0.0001 or not significant (NS) by Mann-Whitney U.

TRIF deficiency results in altered cytokine responses in vivo. (A) Levels of il-6 transcript were measured by RT-qPCR from joint tissue in wild-type (n = 13), TRIF-deficient (n = 13), MyD88-deficient (n = 12), and doubly deficient (DKO) (n = 6) mice from two independent experiments. The lowest value in the wild-type group was arbitrarily set to a value of 1, and all other values are shown in reference to that value using the 2^−ΔΔCT method after normalization for the value of β-actin. Each circle in the data represents one mouse and the lines indicate median values P = 0.0029, P = 0.0002, and P = 0.0033 by Mann-Whitney U. (B) Levels of ifn-β transcript were measured by RT-qPCR from joint tissue in wild-type (WT) (n = 5), TRIF-deficient (n = 7), and MyD88-deficient (n = 4) mice. One mouse in the wild-type group was arbitrarily set to a value of 1, and all other values are shown in reference to that value using the 2^−ΔΔCT method after normalization for the value of β-actin. Each circle in the data represents one mouse and the lines indicate median values. P = 0.0177 and P = 0.0159 by Mann-Whitney U. (C) The levels of il-10 transcript were measured by RT-qPCR from joint tissue in wild-type (WT) (n = 13), TRIF-deficient (n = 14), MyD88-deficient (n = 11), and doubly deficient (DKO) (n = 6) mice. One mouse in the wild-type group was arbitrarily set to a value of 1, and all other values are shown in reference to that value using the 2^−ΔΔCT method after normalization for the value of β-actin. Each circle in the data represents one mouse and the lines indicate median values. P = 0.0088, P = 0.0002, and P = 0.0007 by Mann-Whitney U.