Fig 1.
Macrophages are the main phagocytes to phagocytose leptospires.
(A) Leptospiral phagocytosis in L. interrogans-infected macrophages and neutrophils, determined by confocal microscopy for the indicated infection times. The blue plaques indicate the nucleus. The red spots around the nucleus indicate the intracellular leptospires. (B) Statistical summary of red fluorescence intensity reflecting the intracellular leptospires in L. interrogans-infected macrophages and neutrophils. Statistical data from experiments such as shown in A. Bars show the means ± SD of three independent experiments. Two hundred cells in each experiment were analyzed to quantify the intensities of red fluorescence. The means of red fluorescence intensities from the cells without infection were set as 1.0. *: p<0.05 vs the red fluorescence intensities from the cells without infection. #: p<0.05 vs the red fluorescence intensities from the neutrophils for the indicated times during infection. (C) Leptospiral numbers in L. interrogans-infected macrophages and neutrophils, determined by leptospiral enumeration for the indicated infection times. Bars show the means ± SD of three independent experiments. 106 cells per experiment were used for leptospiral counting. *: p<0.05 vs the leptospiral numbers in the neutrophils for the indicated times during infection. (D) Leptospiral phagosomes in L. interrogans strain Lai-infected macrophages and neutrophils, detected by the transmission electron microscopy. The arrows indicate the intracellular leptospiral phagosomes.
Fig 2.
Macrophages have a higher ability of killing leptospires than neutrophils.
(A) Living and dead leptospires under a confocal microscope. The green leptospires were living while the red leptospires were dead. (B) Ability of macrophages and neutrophils to kill intracellular leptospires during infection with L. interrogans, determined by confocal microscopy for the indicated infection times. Bars show the means ± SD of three independent experiments. Five hundred leptospires in each experiment were analyzed to quantify the values of red fluorescence intensity. The means of red fluorescence intensities from the leptosires without infection were set as 1.0. *: p<0.05 vs the red fluorescence intensities of leptospires without infection. #: p<0.05 vs the red fluorescence intensities reflecting the dead leptospires from the infected Hu- or Ms-neutrophils. ▲: p<0.05 vs the red fluorescence intensities reflecting the dead leptospires from the infected Hu-macrophages. (C) Percentages of dead leptospires from macrophages and neutrophils during infection with L. interrogans, determined by spectrofluorimetry for the indicated infection times. Bars show the means ± SD of three independent experiments. 107 leptospires in each experiment were used to determine the dead leptospiral percentages. *: p<0.05 vs the dead percentages of the leptospires without infection. #: p<0.05 vs the dead leptospiral percentages from the infected Hu- or Ms-neutrophils. ▲: p<0.05 vs the dead leptospiral percentages from the infected Hu-macrophages. (D) Fewer leptospiral colonies from L. interrogans-infected macrophages than neutrophils, assessed by CFU enumeration for the indicated infection times. Bars show the means ± SD of three independent experiments. 106 leptospires from each of the infected cells were serially diluted and then inoculated onto EMJH-agar plates for a three-week incubation at 28°C for CFU enumeration. *: p<0.05 vs the CFU values of the leptospires without infection. #: p<0.05 vs the CFU values of the leptospires from the infected Hu- or Ms-neutrophils. ▲: p<0.05 vs the CFU values of the leptospires from the infected Hu-macrophages. (E) Attenuated growth ability of leptospires from L. interrogans-infected macrophages than neutrophils, assessed by leptospiral enumeration after incubation. Bars show the means ± SD of three independent experiments. 106 leptospires from each of the infected cells were inoculated in EMJH liquid medium for one-week incubation at 28°C for leptospiral enumeration. *: p<0.05 vs the growth ability of the leptospires without infection. #: p<0.05 vs the growth ability of the leptospires from the infected Hu- or Ms-neutrophils. ▲: p<0.05 vs the growth ability of the leptospires from the infected Hu-macrophages. (F) Co-localization of intracellular leptospires with lysosomes under a confocal microscope. Three fluorescence images were merged in the left panels and separate fluorescence channels in the right panel. The blue plaques indicate the nucleus. The green plaques around the nucleus indicate the lysosomes. The red spots around the nucleus indicate the intracellular leptospires. The yellow spots or plaques indicate the co-localization of intracellular leptospires with lysosomes. (G) Co-localization of intracellular leptospires with lysosomes in L. interrogans-infected macrophages and neutrophils, determined by confocal microscopy for the indicated infection times. Bars show the means ± SD of three independent experiments. One hundred cells in each experiment were analyzed to quantify the intensities of yellow fluorescence. The means of yellow fluorescence intensities from the cells without infection were set as 1.0. *: p<0.05 vs the yellow fluorescence intensities of the leptospires without infection. #: p<0.05 vs the yellow fluorescence intensity reflecting the intracellular leptospire-lysosome co-localization in the infected Hu- or Ms-neutrophils. ▲: p<0.05 vs the yellow fluorescence intensity reflecting the intracellular leptospire-lysosome co-localization in the infected Hu-macrophages.
Fig 3.
Higher ROS levels required by macrophages for killing intracellular leptospires.
(A) Total ROS levels in L. interrogans-infected macrophages and neutrophils, determined by confocal microscopy for the indicated infection times. The green fluorescence indicates the intracellular total ROS levels. (B) Statistical summary of green fluorescence intensity reflecting the total ROS levels in L. interrogans-infected macrophages and neutrophils. Statistical data from experiments such as shown in A. Bars show the means ± SD of three independent experiments. Two hundred cells in each experiment were analyzed to quantify the values of fluorescence intensity. The means of fluorescence intensities from the cells without infection were set as 1.0. *: p<0.05 vs the total ROS level in the macrophages and neutrophils without infection. #: p<0.05 vs the total ROS levels in the L. interrogans-infected neutrophils. (C) Lower dead leptospiral percentages in NAC-pretreated L. interrogans-infected Hu-macrophages rather than Hu-neutrophils, determined by spectrofluorimetry for the indicated infection times. Bars show the means ± SD of three independent experiments. 107 leptospires in each experiment were used to determine the dead leptospiral percentages. *: p<0.05 vs the dead leptospiral percentages from the NAC-untreated L. interrogans-infected Hu-macrophages. (D) Lower dead leptospiral percentages in NAC-pretreated L. interrogans-infected Ms-macrophages rather than Ms-neutrophils, determined by spectrofluorimetry for the indicated infection times. The legend is the same as in C except that this experiment was for detection of Ms-macrophages and Ms-neutrophils.
Fig 4.
Higher NO levels and [Ca2+]i are necessary for macrophages to kill intracellular L. interrogans.
(A) Intracellular NO in L. interrogans-infected macrophages or neutrophils under confocal microscope. The green fluorescence indicates the intracellular NO. (B) NO levels in L. interrogans-infected macrophages and neutrophils, determined by confocal microscopy for the indicated infection times. Bars show the means ± SD of three independent experiments. Two hundred cells in each experiment were analyzed to quantify the values of fluorescence intensity. The means of fluorescence intensities from the cells without infection were set as 1.0. *: p<0.05 vs the NO levels in the macrophages and neutrophils without infection. #: p<0.05 vs the NO levels in the L. interrogans-infected neutrophils. (C) Lower dead leptospiral percentages in SMT-pretreated L. interrogans-infected Hu-macrophages rather than Hu-neutrophils, determined by spectrofluorimetry for the indicated infection times. Bars show the means ± SD of three independent experiments. 107 leptospires in each experiment were used to determine the dead leptospiral percentages. *: p<0.05 vs the dead leptospiral percentages from the SMT-untreated L. interrogans-infected Hu-macrophages. (D) Lower dead leptospiral percentages in SMT-pretreated L. interrogans-infected Ms-macrophages rather than Ms-neutrophils, determined by spectrofluorimetry for the indicated infection times. The legend is the same as in C except that this experiment was for detection of Ms-macrophages and Ms-neutrophils. (E) Intracellular free Ca2+ in L. interrogans-infected macrophages or neutrophils under confocal microscope. The green fluorescence indicates the [Ca2+]i. (F) [Ca2+]i in L. interrogans-infected macrophages and neutrophils, determined by confocal microscopy for the indicated infection times. Bars show the means ± SD of three independent experiments. Two hundred cells in each experiment were analyzed to quantify the values of fluorescence intensity. The means of fluorescence intensities from the cells without infection were set as 1.0. *: p<0.05 vs the [Ca2+]i in the macrophages and neutrophils without infection. #: p<0.05 vs the [Ca2+]i in the L. interrogans-infected neutrophils. (G) Lower dead leptospiral percentages in BAPTA/AM-pretreated L. interrogans-infected Hu-macrophages rather than Hu-neutrophils, determined by spectrofluorimetry for the indicated infection times. Bars show the means ± SD of three independent experiments. 107 leptospires in each experiment were used to determine the dead leptospiral percentages. *: p<0.05 vs the dead leptospiral percentages from the BAPTA/AM-untreated L. interrogans-infected Hu-macrophages. (H) Lower dead leptospiral percentages in BAPTA/AM-pretreated L. interrogans-infected Ms-macrophages rather than Ms-neutrophils, determined by spectrofluorimetry for the indicated infection times. The legend is the same as in G except that this experiment was for detection of Ms-macrophages and Ms-neutrophils.
Fig 5.
Histopathological changes and leptospires in tissues of L. interrogans-infected mice.
(A) Histopathological changes in lung, liver and kidney tissues from L. interrogans-infected C3H/HeJ mice, examined by microscopy after HE staining. All the tissues had infiltration of inflammatory cells. The lung, liver and kidney tissues presented serious hemorrhage, extensive hepatocellular necrosis and serious congestion, respectively. (B) Visible leptospires in lung, liver and kidney tissues from L. interrogans-infected C3H/HeJ mice, examined by microscopy after silver staining. The arrows indicate leptospires in the three types of tissues from the infected animals.
Fig 6.
Mononuclear-macrophages from peripheral blood are the main infiltrating phagocytes during leptosirosis.
(A) Infiltration of peripheral blood mononuclear-macrophages and neutrophils into the lung, liver and kidney tissues of L. interrogans-infected C3H/HeJ mice, visualized by immunohistochemistry for the indicated infection times. The mononuclear-macrophages or neutrophils were determined with CD11b or Ly6G antibody. (B) Infiltrated CD11b+ mononuclear-macrophages and Ly6G+ neutrophils in the lung, liver and kidney tissues from L. interrogans-infected mice, estimated by analysis using Image-Pro Plus software. Statistical data from experiments such as shown in A. Bars show the means ± SD of three independent experiments. *: p<0.05 vs the normal animals.
Fig 7.
Profiles of chemokines of mononuclear-macrophages and neutrophils in serum samples from L. interrogans-infected mice and leptospirosis patients.
(A) Detection results of the mouse and human chemokines of mononuclear-macrophages and neutrophils in sera from C3H/HeJ mice during infection with L. interrogans strain Lai, determined by chemokine detection microarrays for the indicated infection times. The spots within red frames indicate the mononuclear-macrophage chemokines, the spots within blue frames indicate the neutrophil chemokines. (B) Statistical summary of the chemokine profile in the serum samples from L. interrogans-infected mice. Statistical data from the microarray detection such as shown in A. Bars show the means ± SD of five independent serum samples. The means of chemokine levels in the serum samples from five mice without infection were set as 1.0. The bars within red frames indicate the mononuclear-macrophage chemokines and the bars within blue frames indicate the neutrophil chemokines. *: p<0.05 vs the chemokine levels in the serum samples from mice without infection. (C) Statistical summary of the chemokine profiles in the serum samples from leptospirosis patients. Statistical data from the microarray detection such as shown in A. Bars show the means ± SD of five patients. The means of chemokine levels in the serum samples from five healthy individuals were set as 1.0. The bars within red frames indicate the mononuclear-macrophage chemokines and the bars within blue frames indicate the neutrophil chemokines. *: p<0.05 vs the chemokine levels in the serum samples from five healthy individuals.
Fig 8.
High expression of VCAM-1, P-selectin or E-selectin in tissues of L. interrogans- infected mice.
(A) Expression of different adhesion molecules of mononuclear-macrophages and neutrophils in the lung, liver and kidney tissues from L. interrogans-infected C3H/HeJ mice, estimated by immunohistochemistry for the indicated infection times. The arrows indicate the expression of different adhesion molecules. (B) Expressed adhesion molecules in the tissues from L. interrogans-infected mice, estimated by analysis using Image-Pro Plus software. Statistical data from experiments such as shown in A. Bars show the means ± SD of three independent experiments. *: p<0.05 vs the normal animals.