Deficiency in the glycosyltransferase Gcnt1 increases susceptibility to tuberculosis through a mechanism involving neutrophils

Abstract

Modulation of immunity and disease by glycans is increasingly recognized. However, how host glycosylation shapes and is shaped by tuberculosis remains poorly understood. We show that deficiency in the glucosaminyl (N-acetyl) transferase 1 (Gcnt1), a key enzyme for core-2 O-glycans biosynthesis, drives susceptibility to Mycobacterium tuberculosis infection. The increased susceptibility of Gcnt1 deficient mice was characterized by extensive lung immune pathology, mechanistically related to neutrophils. Uninfected Gcnt1 deficient mice presented bone marrow, blood and lung neutrophilia, which further increased with infection. Blood neutrophilia required Gcnt1 deficiency in the hematopoietic compartment, relating with enhanced granulopoiesis, but normal cellular egress from the bone marrow. Interestingly, for the blood neutrophilia to translate into susceptibility to M. tuberculosis infection, Gnct1 deficiency in the stroma was also necessary. Complete Gcnt1 deficiency associated with increased lung expression of the neutrophil chemoattractant CXCL2. Lastly, we demonstrate that the transcript levels of various glycosyltransferase-encoding genes were altered in whole blood of active tuberculosis patients and that sialyl Lewis x, a glycan widely present in human neutrophils, was detected in the lung of tuberculosis patients. Our findings reveal a previously unappreciated link between Gcnt1, neutrophilia and susceptibility to M. tuberculosis infection, uncovering new players balancing the immune response in tuberculosis.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1. Deficiency in Gcnt1 associates with increased susceptibility to Mtb infection.

C57BL/6 (B6; black circles) or Gcnt1−/− (open circles) mice were infected by aerosol with Mtb strain HN878 with a low (a, c, e–g) or high (b, d, h, i, g) dose of bacteria. a, b The weight of the animals was monitored to determine survival curves, that included 10–13 animals in two independent experiments. Statistical analysis was performed with a log-rank (Mantel-Cox) test for the Kaplan Meyer curve. c, d At the indicated timepoints post-infection, the lungs of infected mice were collected and the bacteria burden determined by CFU enumeration. e, h At the indicated timepoints post-infection, lung pathology defined as the percentage of infiltrate per lobe (Fig. S1A, B), was determined upon H&E staining and morphometric analysis of the right upper lobes of infected lungs. The histopathologic features of the infected lungs were assessed and a relative score attributed (Table 1); the score obtained for necrosis is plotted in f and i. The pictures in g are H&E staining of representative animals within each experimental group. Arrowheads point to intra-alveolar necrotic debris, black diamond point to bronchiolar debris, black arrows point to perivascular lymphocytes, white arrows point to peribronchiolar lymphocytes and asterisks to calcification sites. Scale bar corresponds to 100 µm. In c each dot represents the Mean ± SEM and in d–h each dot represents an individual mouse of 6–12 in at least two independent experiments. In e, f red dots represent moribund Gcnt1−/− mice. Statistical analysis was performed using multiple t-test (c) or unpaired t-test (d–i). *p < 0.05; **p < 0.01; ***p < 0.01; ****p < 0.0001.

Fig. 2. Exacerbated neutrophilia drives increased susceptibility of Gcnt1−/− mice to Mtb infection.

a–e At the indicated timepoints post-infection, the lungs of C57BL/6 (B6; black circles) or Gcnt1−/− (open circles) mice infected by aerosol with a low or high dose of Mtb strain HN878 were harvested and a cellular suspension prepared. a, b, e The relative expression of the indicated genes was determined by real-time PCR. c Percentages of the indicated immune cell populations were determined by flow cytometry. The gating strategy is shown in Fig. S2A. d Representative images of MPO staining (green) in lung sections of B6 and Gcnt1−/− mice infected with low or high doses of Mtb. Sections were counterstained with DAPI (blue). Scale bars correspond to 1 mm (left panels) and 100μm (right panels). In a, c each dot represents the Mean ± SEM for 10 animals in two independent experiments. Non-infected animals (day 0) were used as controls. b, e Represented are heatmaps of log2 relative expression of the indicated genes in lung samples of B6 and Gcnt1−/− mice 27 days after high dose Mtb infection. f C57BL/6 (B6, black circles) or Gcnt1−/− mice were infected by aerosol with a high dose of Mtb strain HN878. On day 18 post-infection the Gcnt1−/− mice were treated with the neutrophil-depleting anti-Ly6G mAb (black triangles) or an isotype control (open circles). The weight of the animals (5 per group) was monitored to determine survival curves. Statistical analysis was performed per time point with unpaired two-tailed Mann-Whitney test (a, c) or with log-rank (Mantel-Cox) test for the Kaplan Meyer curve (f). * refer to statistic differences between C57BL/6 or Gcnt1−/− mice. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 3. Gcnt1 modulates granulopoiesis and CXCL2 expression.

The BM of non-infected C57BL/6 (B6; black circles) or Gcnt1−/− (open circles) mice was harvested and the frequency of a LSK, CMP and GMP populations; b neutrophils and monocytes or (c) neutrophils expressing CXCR4 determined by flow cytometry. All gating strategies are shown in Fig. S3. d Egress of immune cell populations from the BM of in C57BL/B6 (B6; black circles) or Gcnt1−/− (open circles) measured as the percentage of CD45+ BM sinusoidal cells upon intravenous injection of CD45-PE. e Permeability of the lung vasculature in C57BL/B6 (B6; black circles) or Gcnt1−/− (open circles) measured through quantification of the amount of Evans Blue extravasated into the tissue upon intravenous injection. f The expression of Cxcl1, Cxcl2 and Cxcl5 in the lungs of mice infected with low or high doses of Mtb was determined by real-time PCR, for the indicated timepoints post-infection. g Analysis of neutrophils migration through 5 μm transwells towards different concentrations of recombinant CXCL1, CXCL2 and CXCL5. Mean ± SEM of culture triplicates are presented. In a, f each dot represents a mouse and the Mean±SEM for 6–15 animals in at least two independent experiments are plotted. Statistical analysis was performed with Student’s t-test (a–f) or unpaired two-tailed Mann-Whitney test for each time point (g). * refer to statistic differences between B6 or Gcnt1−/− mice. *p < 0.05; **p < 0.01; ***p < 0.01; ****p < 0.0001.

Fig. 4. Blood neutrophilia of Gcnt1−/− mice is promoted by deficiency of this enzyme in hematopoietic cells, but increased susceptibility to Mtb infection also requires the non-hematopoietic compartment.

a Schematic representation of the BM transplantation model used and the experimental groups included. b The frequency of neutrophils in the blood of non-infected chimeric mice was determined by flow cytometry, following the gating strategy shown in Fig. S3. c Mice in the different chimeric groups were infected with a low dose of Mtb strain HN878 and the weight of the animals monitored to determine survival curves. d On day 30 post-infection, the bacterial burden in the lungs of the infected mice was determined by CFU enumeration. e H&E staining of one representative animal of each experimental group is represented. Black arrows point to edema, black arrowhead spot bronchopneumonia, and dashed black line limits the necrotic areas. Scale bar corresponds to 100 µm. f The number of neutrophils present in infected lungs was determined on day 30 post-infection, by flow cytometry. g The expression of Cxcl1, Cxcl2 and Cxcl5 in the lungs of infected mice was determined by real-time PCR, as above. b, d, f, g Represented is Mean±SEM, and each symbol represents one mouse. Statistical analysis was performed with a one-way ANOVA using Tukey’s test for multiple comparisons (b, d, f, g) or with log-rank (Mantel-Cox) test for the Kaplan Meyer curve (c). * refer to statistic differences between the indicated chimeric groups. **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 5. Mtb infection impacts the expression of several glycosyltransferase-encoding genes in humans.

a Neutrophils are the main cells expressing sLex in human peripheral blood. Vein blood was collected and stained for CD3, CD14, CD16, CD19 and sLex by flow cytometry. Data shown represent one donor out of 25 analysed. b Volcano plots displaying the comparisons between Active TB vs Control, Active TB vs Latent TB and Latent TB vs Control regarding the differential expression of annotated coding genes of glycosyltransferases (obtained from Glyco-Enzyme Repository) on the Berry London, Berry Leicester (progressor) and Berry South Africa datasets. Labelled genes encode for glycosyltransferases associated with the sLex pathway. Dot color indicates if the corresponding gene is upregulated (red, log-fold-change >1 and p-value < 0.05), downregulated (blue, log-fold-change < -1 and p-value < 0.05), significant (black, log-fold-change >−1 to <1 and p-value < 0.05) and not significant (grey). c Lung sections of TB patients who underwent therapeutic surgery were stained for sLex as above. Black and white arrowheads point to positive epithelial or immune cells, respectively. Scale bars correspond to 100 µm.