Two-Photon Intravital Imaging of Lungs during Anthrax Infection Reveals Long-Lasting Macrophage-Dendritic Cell Contacts

Abstract

eThe dynamics of the lung immune system at the microscopic level are largely unknown because of inefficient methods of restraining chest motion during image acquisition. In this study, we developed an improved intravital method for two-photon lung imaging uniquely based on a posteriori parenchymal tissue motion correction. We took advantage of the alveolar collagen pattern given by the second harmonic generation signal as a reference for frame registration. We describe here for the first time a detailed dynamic account of two major lung immune cell populations, alveolar macrophages and CD11b-positive dendritic cells, during homeostasis and infection by spores of Bacillus anthracis, the agent of anthrax. We show that after alveolar macrophages capture spores, CD11b-positive dendritic cells come in prolonged contact with infected macrophages. Dendritic cells are known to carry spores to the draining lymph nodes and elicit the immune response in pulmonary anthrax. The intimate and long-lasting contacts between these two lines of defense may therefore coordinate immune responses in the lung through an immunological synapse-like process.T he lung is a crucial and yet very delicate organ dedicated to gas exchange through a thin alveolar-capillary wall (1). The obscure side of this highly efficient system is the constitutive fragility of its extensive exchange surfaces. The tour de force of the lungassociated immune system consists of its innate ability to protect against infection and pollutants while maintaining an anti-inflammatory environment (2). This complicated balance is achieved by a network of scattered cells such as alveolar macrophages on the outer side of the alveoli and different subsets of interstitial dendritic cells (DCs) (3).Recent advances in intravital microscopy (IVM) using twophoton excitation fluorescence (TPEF) have profoundly improved our understanding of the immune system dynamics in most organs, except the lungs (4). A detailed account of hostpathogen interactions in diverse tissues has been given (5-11), but lung immune system investigation by IVM has lagged due to the lack of a noninvasive and effective method taking into account lung features.Recent studies have addressed rigorous lung parenchyma IVM using TPEF in inflammation models (12). Two alternative a priori stabilization methods have been used by others to restrain movement compatible with imaging, based either on gluing the parenchyma onto a frame (13) or on a suction system stabilizing the lung under a glass window (14). These techniques, although effective for various purposes, are not optimal, as they alter the physiological motion of the parenchyma and may provoke unexpected reactions.We describe here an alternative approach for studying the lung by IVM based uniquely on a posteriori tissue motion correction. We take advantage of the fibrillar collagen pattern given by second harmonic generation (SHG) signals as a reference for frame registration and removal of wobbling images. This method allows acquisition in two spatial dime...

Intravital microscopy of the lung: minimizing invasiveness

“…The animal preparation prior to imaging is performed according to this consensual protocol [11,18,[23][24][25][26][27][28][29]:…”

“…While mouse physiology requires a breathing frequency of 106 bpm to 230 bpm, depending upon other factors such as strain and weight [57], it is possible to modulate this frequency to 130 bpm [28], 120 bpm [11,27,29], 100 bpm [25], or even 60 bpm for the recording period after which ventilation is returned to 100 bpm [26]. This decrease of the breathing frequency noticeably increases stability of the images.…”

“…We have recently used this technique to analyze lung macrophage and dendritic cell dynamics, demonstrating that bacterial spores enhance in vivo the contacts between these two immune cell populations, possibly facilitating antigen transfer (Figure 2e) [11].…”

“…Thus, T −1 is a rotation matrix. Parameters of these matrices can be determined by cross correlation with a reference image that is a biological structure stable over-time [11,16].…”

“…The second section of this review describes these very techniques, beginning with pioneers such as Malpighi who intellectually filtered his unstable observation before drawing the resulting image, to computerized post-processing strategies based on frame registration of oversampled acquisition through mechanically-stabilized organ. These latter techniques have recently opened a new exciting window upon the dynamic study of the pulmonary immune system [6][7][8][9][10][11][12].…”