Adipose tissue is the first colonization site of <i>Leptospira interrogans</i> in subcutaneously infected hamsters

<div><p>Leptospirosis is one of the most widespread zoonoses in the world, and its most severe form in humans, “Weil’s disease,” may lead to jaundice, hemorrhage, renal failure, pulmonary hemorrhage syndrome, and sometimes,fatal multiple organ failure. Although the mechanisms underlying jaundice in leptospirosis have been gradually unraveled, the pathophysiology and distribution of leptospires during the early stage of infection are not well understood. Therefore, we investigated the hamster leptospirosis model, which is the accepted animal model of human Weil’s disease, by using an <i>in vivo</i> imaging system to observe the whole bodies of animals infected with <i>Leptospira interrogans</i> and to identify the colonization and growth sites of the leptospires during the early phase of infection. Hamsters, infected subcutaneously with 10⁴ bioluminescent leptospires, were analyzed by <i>in vivo</i> imaging, organ culture, and microscopy. The results showed that the luminescence from the leptospires spread through each hamster’s body sequentially. The luminescence was first detected at the injection site only, and finally spread to the central abdomen, in the liver area. Additionally, the luminescence observed in the adipose tissue was the earliest detectable compared with the other organs, indicating that the leptospires colonized the adipose tissue at the early stage of leptospirosis. Adipose tissue cultures of the leptospires became positive earlier than the blood cultures. Microscopic analysis revealed that the leptospires colonized the inner walls of the blood vessels in the adipose tissue. In conclusion, this is the first study to report that adipose tissue is an important colonization site for leptospires, as demonstrated by microscopy and culture analyses of adipose tissue in the hamster model of Weil’s disease.</p></div

<i>Leptospira</i> distribution in skin and subcutaneous tissue.

<p>Representative light field (A, C) and fluorescence images (B, D) of the skin and subcutaneous tissue (A, B) or adipose tissue (C, D) around the injection sites of M1307 collected from infected hamsters at phase 4. Fluorescence images (B, D) showing cell nuclei stained with DAPI (blue), autofluorescence of the skin and subcutaneous tissue (green, not shown in panel D), and leptospires stained with rabbit polyclonal antiserum and Cy5-conjugated anti-rabbit monoclonal antibody (red). The framed area in (B) is enlarged at the upper right. Scale bars: 100 μm (A, B), 500 μm (C, D).</p

Bioluminescence dissemination of <i>Leptospira</i> in hamsters.

<p>(A) The survival rate of Golden Syrian hamsters (n = 8) infected subcutaneously with 10⁴ <i>L</i>. <i>interrogans</i> strain M1307 into the right inguinal region, and representative ventral view photographic images tracking the hamster infections on different days post-infection. Images depict photographs overlaid with color representations of luminescence intensity, measured in photons/second/cm²/sr as indicated on the scales, where red is the most intense (3×10⁵) and purple is the least intense (3×10⁴). (B,C) Average luminescence intensities in each ROI of injection site (B) and abdominal center (C) at different days post-infection. Data are expressed as the means ± SEM of total flux in photons/second in each ROI in eight infected hamsters (●) and two uninfected controls (◦). <i>p</i> values (*<i>p</i><0.05), between groups.</p

Bioluminescence changes in hamster organs.

<p>Representative bioluminescence images (ventral view) from M1307-infected hamsters at each phase. Images represent subcutaneous tissues after skin incision and organs after laparotomy, as well as <i>ex vivo</i> organs (blood plus liver and kidney cross sections). The scale is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172973#pone.0172973.g001" target="_blank">Fig 1</a>.</p

Transmission electron microscopy of adipose tissue blood vessels.

<p>Representative transmission electron microscope images of subcutaneous adipose tissue blood vessels around the injection sites of <i>Leptospira</i>-infected hamsters at phase 4. The framed area in (A) is enlarged in (B). The scale bars represent 5 μm (A) and 1 μm (B). The arrowheads point to <i>Leptospira</i> and the arrows show the red blood cells.</p

Transplantation of HSCs and hematopoietic progenitors.

<p>(A) LSKs or CD34⁻LSKs were purified by FACS from Lin^−/lowc-Kit⁺ BM fraction of GFP mice. GFP and Lineage+propidium iodide (PI) expression of unfractionated BM cells is shown as a control. (B) Total myeloid progenitors (My-P) and CLPs were purified by FACS from Lin^−/lowThy1.2^−/lowMNCs of GFP mice. (C-E) Correlational analyses between injected cell numbers and the numbers of GFP⁺ cardiomyocytes per a recipient mouse in recipients transplanted with Lin^−/lowSca-1⁺ cells (C), LSKs (D), and CD34⁻LSKs (E). (F) Comparison of the number of GFP⁺ cardiomyocytes at the same injected cell dose in Lin^−/lowSca-1⁺ cells, LSKs, and CD34⁻LSKs recipients. The means of the number of GFP⁺ cardiomyocytes in recipients transplanted with each injected cell number are plotted. In the transplantation of LSKs, injected cell number of 1–1.8×10⁵ cells is plotted at 10⁵, that of 1–4×10⁴ cells is plotted at 10⁴, and that of 1–2×10³ cells is plotted at 10³. The greater cardiomyogenic ability existed in CD34⁻LSKs than LSKs, and in LSKs than Lin^−/lowSca-1⁺ cells.</p

Characterization of donor BM-derived GFP⁺ cells in injured heart.

<p>(A) Representative image of CD11b-expressing GFP⁺ myeloid cells. GFP⁺ hematopoietic cells in recipient cardiac tissue appeared in small and round shape. Cardiac section was stained with anti-CD11b (red, Cy3) and DAPI (blue). Inset: high magnification of GFP⁺CD11b⁺ cells. (B) Representative image of a vimentin-expressing GFP⁺ fibroblast. Cardiac section was stained with anti-vimentin (red, Cy3). The fibroblast was present adjacent to striated cardiomyocytes in differential interference contrast (DIC) image. (C and D) Representative images of a TnI- (C) or Cx43- (D) expressing GFP⁺ striated cardiomyocyte. Cardiac sections were stained with anti-TnI (C; red, Cy3), anti-Cx43 (D; yellow, Cy5), and DAPI (blue). Cardiac sections are from recipients transplanted with unfractionated BM cells. All merged images were obtained from the same confocal plane. Scale bars = 50 µm, (A)-inset 10 µm.</p

The number of donor-derived cardiomyocytes after transplantation of purified BM cells.

a<p>P = 0.0095 by Mann-Whitney U test, Lin^−/lowCD45⁺ versus Lin^−/lowCD45^−.</p>b<p>P = 0.0021 by Mann-Whitney U test, Myeloid progenitor versus CLP.</p>c<p>P = 0.0004 by Mann-Whitney U test, CMP versus CLP.</p>d<p>P = 0.1142 by Mann-Whitney U test, Myeloid progenitor versus CLP.</p>*<p>GFP⁺ cardiomyocytes were counted in 40 contiguous sections from apex of the heart per a mouse. Detailed information of histological analysis is described in Materials and Methods, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062506#pone.0062506.s008" target="_blank">Materials and Methods S1</a>.</p><p>Abbreviations. BM: bone marrow, PB: peripheral blood, HSC: hematopoietic stem cell, LSK: Lin⁻Sca-1⁺c-Kit⁺, CMP: common myeloid progenitor, GMP: granulocyte/monocyte progenitor, CLP: common lymphoid progenitor.</p