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. 2013 Apr;5(4):516-30.
doi: 10.1002/emmm.201202023.

A novel murine model of rhinoscleroma identifies Mikulicz cells, the disease signature, as IL-10 dependent derivatives of inflammatory monocytes

Cindy Fevre  1 , Ana S Almeida, Solenne Taront, Thierry Pedron, Michel Huerre, Marie-Christine Prevost, Aurélie Kieusseian, Ana Cumano, Sylvain Brisse, Philippe J Sansonetti, Régis Tournebize
Affiliations

A novel murine model of rhinoscleroma identifies Mikulicz cells, the disease signature, as IL-10 dependent derivatives of inflammatory monocytes

Cindy Fevre et al. EMBO Mol Med. 2013 Apr.

Abstract

Rhinoscleroma is a human specific chronic disease characterized by the formation of granuloma in the airways, caused by the bacterium Klebsiella pneumoniae subspecies rhinoscleromatis, a species very closely related to K. pneumoniae subspecies pneumoniae. It is characterized by the appearance of specific foamy macrophages called Mikulicz cells. However, very little is known about the pathophysiological processes underlying rhinoscleroma. Herein, we characterized a murine model recapitulating the formation of Mikulicz cells in lungs and identified them as atypical inflammatory monocytes specifically recruited from the bone marrow upon K. rhinoscleromatis infection in a CCR2-independent manner. While K. pneumoniae and K. rhinoscleromatis infections induced a classical inflammatory reaction, K. rhinoscleromatis infection was characterized by a strong production of IL-10 concomitant to the appearance of Mikulicz cells. Strikingly, in the absence of IL-10, very few Mikulicz cells were observed, confirming a crucial role of IL-10 in the establishment of a proper environment leading to the maturation of these atypical monocytes. This is the first characterization of the environment leading to Mikulicz cells maturation and their identification as inflammatory monocytes.

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Figures

Figure 1
Figure 1
Colonization and histology of BALB/c lungs infected by K. rhinoscleromatis. A. Bacterial load in lungs of mice infected with 2.107 K. rhinoscleromatis (left), 2.107 Kp52.145 (centre) or 2.104 Kp52.145 (right). Data show log CFU/organ from 5 to 23 mice. Means are indicated as line. B. Quantification of the number of cells present in lungs of saline-injected mice, mice infected with 2.107 K. rhinoscleromatis (KR) or 2.104 Kp52.145 (KP). C–F. Lungs of mice infected by 2.107 K. rhinoscleromatis were resected 1, 3 and 5 days post-infection and observed by histology. One day post-infection (C) lungs presented a moderate inflammation with intact alveoli containing few alveolar macrophages (inset top) and numerous bacilli (inset bottom). Three days post-infection (D) non-specific inflammatory lesions increased and a few spumous histiocytes appeared (inset). Five days post-infection (E) two types of inflammatory lesions could be seen and distinguished based on their brightness. The right of the picture is composed of darker features that corresponded to classical abscessed lesions with numerous polymorphonuclear cells. The middle and left part of this panel were brighter and corresponded to the reaction implicating foamy histiocytes. A zoom into this region (F) revealed intact alveoli filled exclusively with big spumous histiocytes. Scale bars: C, D and E, 100 µm; insets and F, 10 µm. G. Ultrastructural features of lungs of mice after 4 days of infection with K. rhinoscleromatis. Lungs revealed three types of Mikulicz cells, which seem to correspond to three different maturation steps. Mikulicz cells first presented few individual vacuoles (bottom cell) that tended to fuse during maturation (middle and inset) before forming a single enlarged vacuole (top right). Scale bars: 2 µm.
Figure 2
Figure 2
Kinetics of cells recruitment after infection with K. rhinoscleromatis and Kp52.145. A. Lung cells of BALB/c mice infected with 2.107 K. rhinoscleromatis, 2.104 Kp52.145 or saline-injected controls were isolated 1, 3 and 5 days post inoculation and stained for granulocytes (Gr1+ F4/80− CD11b+ CD11c−), resident monocytes (Gr1− F4/80+ CD11b+ CD11c−), alveolar macrophages (Gr1- F4/80+ CD11b− CD11c+) or inflammatory monocytes (Gr1+ F4/80+ CD11b+ CD11c−). Results show the percentage of each cell population among the total lung cells. Data are mean ± SEM and represent between 6 and 12 mice for each point from at least three independent experiments. B. Forward scatter histogram of resident monocytes (RM, red) and inflammatory monocytes (IM, blue). C. Quantification of sorted inflammatory monocytes size. Cell size was measured 3 (white bars, n = 49) and 5 (black bars, n = 119) days post-infection. D. Three and five days post-infection by K. rhinoscleromatis, cells were isolated and the inflammatory monocyte population was sorted by FACS, centrifuged onto slides and stained with HE. Classical monocytes (left) and small Mikullicz cells (middle) were observed 3 days post-infection. Large Mikulicz cells (right) were present 5 days post infection. Scale bar: 10 µm.
Figure 3
Figure 3
CCR2-independent recruitment of Mikulicz cells from the bone marrow to the lungs upon K. rhinoscleromatis infection. A. Expression of CD45.1 and CD45.2 markers on gated inflammatory monocytes from non-irradiated F1 (C57BL/6;BALB/c, parent), CD45.1-expressing C57BL/6 (donor) and irradiated and reconstituted F1 (recipient) infected with K. rhinoscleromatis. Graphs show representative data of one mouse out of ten infected recipient mice from two independent experiments. B. Number of inflammatory monocytes in lungs from CCR2−/− and C57BL/6 WT mice 3 days post-infection with K. rhinoscleromatis. Data are mean ± SEM from 9 to 10 mice per group from two independent experiments. C. Typical Mikulicz cells are present in lungs from infected C57BL/6 WT or CCR2−/− mice (arrows). Scale bar: 10 µm. D. Quantification of Mikulicz cells in lung sections of C57BL/6 WT or CCR2−/− mice. Data are mean ± SEM. n = 10.
Figure 4
Figure 4
Production of IL-1β, IL-10 and IL-17 in the lungs of mice infected with K. rhinoscleromatis, Kp52.145 or Kp110. A. BALB/c mice were injected with saline or infected with 2.107 K. rhinoscleromatis or 2.104 Kp52.145 for 1, 3 or 5 days. Lungs were homogenized and cytokines were measured in the extracts by ELISA. Data are mean ± SEM from 8 to 15 mice from two independent experiments. B. Mice were injected with saline or infected with 2.107 K. rhinoscleromatis, Kp52.145 or Kp110 strains. Cytokines were measured at different days post infection (1, 2, 3 and 5 for K. rhinoscleromatis; 1, 3 and 5 for Kp110; 1 and 2 for Kp52.145). Data are mean ± SEM from 3 to 9 mice from two independent experiments.
Figure 5
Figure 5
Absence of Mikulicz cells in IL10−/− mice. A. Percentage of inflammatory monocytes in lungs from IL-10−/− and BALB/c WT mice 3 days post-infection with 106 K. rhinoscleromatis. (*p = 0.04). Data are representative of 7 to 10 mice from three independent experiments. B. Forward scatter histogram of inflammatory monocytes in WT (blue) and IL10−/− (red) mice. Cells are smaller in IL10−/− mice. C. Comparison of number of Mikulicz cells in tissue sections between BALB/c WT and IL10−/− mice. Data are mean ± SEM. n = 16 and 38. (***p < 0.0001). D. Histology, representative examples of lungs in IL10−/− and BALB/c WT mice. Inset show Mikulicz cells in WT and granulocytes and polymorphonuclear cells in IL10−/− mice. Scale bars 100 µm, inset 10 µm.
Figure 6
Figure 6
Blocking IL-10R leads to the absence of Mikulicz cells. A. Percentage of inflammatory monocytes in lungs of BALB/c mice treated with IL-10R blocking antibody or control IgG 4 days post-infection with K. rhinoscleromatis. (***p < 0.001). Data are representative of 10 mice from two independent experiments. B. Comparison of number of Mikulicz cells in tissue sections between BALB/c WT mice and mice injected with IL10R neutralizing antibody. Data are mean ± SEM. n = 39 and 40. ***p < 0.0001. C. Histology, representative examples of lungs in mice injected with IL-10R neutralizing antibody or control IgG. Arrow points to a rare Mikulicz cell observed. Scale bars: 100 µm.
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