Mycobacterial Infection of Precision-Cut Lung Slices Reveals Type 1 Interferon Pathway Is Locally Induced by Mycobacterium bovis but Not M. tuberculosis in a Cattle Breed

Abstract

Tuberculosis exacts a terrible toll on human and animal health. While Mycobacterium tuberculosis (Mtb) is restricted to humans, Mycobacterium bovis (Mb) is present in a large range of mammalian hosts. In cattle, bovine TB (bTB) is a noticeable disease responsible for important economic losses in developed countries and underestimated zoonosis in the developing world. Early interactions that take place between mycobacteria and the lung tissue early after aerosol infection govern the outcome of the disease. In cattle, these early steps remain poorly characterized. The precision-cut lung slice (PCLS) model preserves the structure and cell diversity of the lung. We developed this model in cattle in order to study the early lung response to mycobacterial infection. In situ imaging of PCLS infected with fluorescent Mb revealed bacilli in the alveolar compartment, in adjacent or inside alveolar macrophages, and in close contact with pneumocytes. We analyzed the global transcriptional lung inflammation signature following infection of PCLS with Mb and Mtb in two French beef breeds: Blonde d'Aquitaine and Charolaise. Whereas, lungs from the Blonde d'Aquitaine produced high levels of mediators of neutrophil and monocyte recruitment in response to infection, such signatures were not observed in the Charolaise in our study. In the Blonde d'Aquitaine lung, whereas the inflammatory response was highly induced by two Mb strains, AF2122 isolated from cattle in the UK and Mb3601 circulating in France, the response against two Mtb strains, H37Rv, the reference laboratory strain, and BTB1558, isolated from zebu in Ethiopia, was very low. Strikingly, the type I interferon pathway was only induced by Mb but not Mtb strains, indicating that this pathway may be involved in mycobacterial virulence and host tropism. Hence, the PCLS model in cattle is a valuable tool to deepen our understanding of early interactions between lung host cells and mycobacteria. It revealed striking differences between cattle breeds and mycobacterial strains. This model could help in deciphering biomarkers of resistance vs. susceptibility to bTB in cattle as such information is still critically needed for bovine genetic selection programs and would greatly help the global effort to eradicate bTB.

Keywords: Mycobacterium bovis; alveolar macrophages; cattle; ex vivo; precision cut lung slices; type I interferon.

Figure 1

Precision-cut lung slice (PCLS) infection with four different Mb or Mtb strains does not induce lung tissue cytotoxicity, and equivalent numbers of bacilli are recovered 24 h post-infection. (A) PCLS prepared from Blonde d'Aquitaine lungs post-mortem were infected with 10⁵ CFU of two Mb strains (AF2122 or Mb3601) or two Mtb strains (H37Rv or BTB1558). After 1 and 2 days post-infection, the PCLS supernatants were harvested, and tissue was homogenized. Lactate dehydrogenase (LDH) was measured in both compartments using the “non-radioactive cytotoxicity assay” kit. Cytotoxicity was determined as (%) = (O.D. 490 nm LDH in supernatant)/(O.D. 490 nm LDH in supernatant + O.D. 490 nm LDH in PCLS homogenates) × 100. Individual data and the median and interquartile range in each group are presented (n = 6 animals from six independent experiments). (B) At 24 h post-infection, the PCLS were washed and homogenized to recover bacilli. The inoculum and PCLS homogenates were serially diluted and plated with colony-forming units numerated after 3–6 weeks of incubation. Individual data and the mean in each group are presented (n = 6 independent inocula prepared; PCLS homogenates data represent the mean of technical duplicates from n = 3 animals from three independent experiments).

Figure 2

Mb3601 is internalized by alveolar macrophages (AMPs) in the preserved lung structure from precision-cut lung slice (PCLS), and the infected alveoli contain higher numbers of AMPs compared to non-infected alveoli. The PCLS were infected with 10⁵ colony-forming units of the green fluorescent protein Mb3601-GFP recombinant strain and fixed 2 days later. After labeling with anti-pancytokeratine (magenta) and anti-MHCII antibodies (Alexa 555, red), the PCLS were mounted with Fluoromount-G™ mounting medium containing DAPI (blue) and analyzed under a Leica confocal microscope (A); 3D images were analyzed with Leica LAS software. Z-stack imaging was performed at × 63 enlargement (10–15 μm in thickness, step size of 0.5–1 μm). The white asterisks indicate extracellular bacilli, and the white arrows indicate bacilli inside MHC-II^pos AMPs. (B) The graph represents the percentage of infected alveoli per PCLS among the 55–80 alveoli that were observed under the microscope (n = 4 PCLS from two different Blonde d'Aquitaine cattle). (C) Stack histogram of the mean percentage ± SEM of intra- or extracellular bacilli among a minimum of 15 infected alveoli that were observed (N = 4 PCLS). (D) The number of MHC-II^pos AMPs per alveoli was counted in infected or non-infected alveoli. The data presented as percent are the mean ± SEM of n = 4 PCLS from two different Blonde d'Aquitaine cattle. Between 55 and 80 alveoli were observed to obtain these data (two-way ANOVA, ***p < 0.001).

Figure 3

Principal component analysis (PCA) of inflammatory lung tissue signature reveals differences between two beef cattle breeds after 2 days of infection by Mb or Mtb. (A) Fifteen cytokines and chemokines were measured in PCLS supernatants from Blonde d'Aquitaine or Charolaise cows 2 days after infection with four different mycobacterial strains. Raw data were used to run PCA in R studio. Individual data are shown (n = 4 for Charolaise, red; n = 6 for Blonde d'Aquitaine, blue). The ellipses represent a confidence range of 90%. (B) PCA were built from the expression data of 96 genes (2^−ΔΔCt) obtained from precision-cut lung slice total RNA extracted 2 days after infection. Individual data are shown (n = 9 for Charolaise, red; n = 7 for Blonde d'Aquitaine, blue). The ellipses represent a confidence range of 90%. (C) Two examples of differentially expressed genes. Individual data and the median and interquartile range in each group are presented (n = 7 Blonde d'Aquitaine and n = 9 Charolaise) *p < 0.05; **p < 0.01; ***p < 0.001. Two-way ANOVA test.

Figure 4

The lung inflammatory neutrophil and monocyte recruitment signature induced by infection in precision-cut lung slice (PCLS) from Blonde d'Aquitaine cows is more efficiently triggered by Mycobacterium bovis than Mycobacterium tuberculosis. (A) The cytokine and chemokine levels were measured in PCLS supernatant by Multiplex ELISA 2 days after infection with two Mb or two Mtb strains. Individual data and the median and interquartile range in each group are presented (n = 6 cows). (B) Table of the mean of fold change (2^−ΔΔCT) for each group (n = 7 cows) of 17 major genes involved in neutrophil and monocyte recruitment and inflammation. The graduated red box coloring represents levels of gene expression, and the asterisks mark significant differences compared to non-infected controls. (C) CXCL2, CXCL5, and CXCL8 gene expression at 2 days post-infection. Individual data and the median and interquartile range in each group are presented (n = 7 cows). (B,C) *p < 0.05 (Wilcoxon nonparametric test).

Figure 5

Mb but not Mtb infection in the lung tissue from Blonde d'Aquitaine cows induces the type I interferon pathway. The precision-cut lung slice (PCLS) was infected as described in Figure 1. (A) IFNAR, ISG15, CXCL10, and OAS1 gene expression at 2 dpi. Individual data and the median and interquartile range in each group are presented (n = 7). (B) CXCL10 protein level was measured in PCLS supernatant at 2 dpi. Individual data and the median and interquartile range in each group are presented (n = 6). (C) The table represents the mean of fold change (2^−dCT) for each group (n = 7) of major genes involved in type I interferon pathway. The graduated red box coloring is for higher gene expression, and the asterisks mark significant differences compared to uninfected PCLS. nd, not detected. (D) Ingenuity pathway analysis drawing of the type I interferon pathway under IFNAR in the Mb3601 group. The graduated red box coloring is for higher gene expression. (A–C) *p < 0.05 (Wilcoxon nonparametric test).

Figure 6

Alveolar macrophages (AMP) from Blonde d'Aquitaine contribute to the type I IFN signature in the lung induced by Mb infection. AMPs from Blonde d'Aquitaine lungs were infected with 10⁵ CFU of Mb3601 or Mtb H37Rv. At 6 h later, mRNA was extracted, and the expression of major genes from the type 1 IFN pathway was analyzed. (A) Mean fold change (2^−ΔΔCT) of gene expression normalized to three housekeeping genes was calculated in each group (n = 7). The graduated red box coloring represents gene expression, and the asterisks mark significant differences compared to non-infected controls (nd, not detected). (B) IFNβ, LPG2, RIG1, MX1, and OAS1 gene expression in AMPs was analyzed by RT-qPCR at 6 h post-infection. Individual data and the median and interquartile range in each group are presented (n = 7). (A,B) *p < 0.05 (Wilcoxon nonparametric test).

Figure 7

Superposition of Blonde d'Aquitaine and Charolaise beef breeds in French counties where Mb outbreaks were declared between December 2019 and 2020. This map of France shows the counties where Mb outbreaks were declared between December 2019 and December 2020 (yellow stars) and was obtained with data extracted from https://www.plateforme-esa.fr/. Herd densities of Blonde d'Aquitaine (blue), Charolaise (red), or both breeds (violet) were extracted from data obtained from https://www.racesdefrance.fr/ (cows above 3 years old have been considered).