Macrophage Galactose-Type Lectin-1 Deficiency Is Associated with Increased Neutrophilia and Hyperinflammation in Gram-Negative Pneumonia

Abstract

C-type lectin receptors (CLRs), the carbohydrate-recognizing molecules, orchestrate host immune response in homeostasis and in inflammation. In the present study we examined the function of macrophage galactose-type lectin-1 (MGL1), a mammalian CLR, in pneumonic sepsis, a deadly immune disorder frequently associated with a nonresolving hyperinflammation. In a murine model of pneumonic sepsis using pulmonary infection with Klebsiella pneumoniae, the expression of MGL1 was upregulated in the lungs of K. pneumoniae-infected mice, and the deficiency of this CLR in MGL1(-/-) mice resulted in significantly increased mortality to infection than in the MGL1-sufficient wild-type mice, despite a similar bacterial burden. The phagocytic cells from MGL1(-/-) mice did not exhibit any defects in bacterial uptake and intracellular killing and were fully competent in neutrophil extracellular trap formation, a recently identified extracellular killing modality of neutrophils. Instead, the increased susceptibility of MGL1(-/-) mice seemed to correlate with severe lung pathology, indicating that MGL1 is required for resolution of pulmonary inflammation. Indeed, the MGL1(-/-) mice exhibited a hyperinflammatory response, massive pulmonary neutrophilia, and an increase in neutrophil-associated immune mediators. Concomitantly, MGL1-deficient neutrophils exhibited an increased influx in pneumonic lungs of K. pneumoniae-infected mice. Taken together, these results show a previously undetermined role of MGL1 in controlling neutrophilia during pneumonic infection, thus playing an important role in resolution of inflammation. To our knowledge, this is the first study depicting a protective function of MGL1 in an acute pneumonic bacterial infection.

Figure 1

MGL1 is upregulated in the lungs of Klebsiella pneumoniae (KPn) infected pneumonic mice. (A) Total RNA was extracted by Trizol method from the lungs of KPn infected wild-type (WT) C57/BL6 mice, harvested at indicated times post-infection. The mRNA levels of MGL1 were analyzed by real-time PCR as described in Methods and are expressed as fold changes over the levels in mock control mice calculated by using the formula 2^−(ΔΔCt). Data shown are the mean ± SEM of 3–4 mice per time point in two independent experiments. Significant differences were measured by Student’s t test (p < 0.05) (B) MGL1 expression was examined by immunofluorescence staining on lung cryosections of mock control and KPn infected wild-type mice harvested at indicated times post-infection using an affinity purified anti-mouse MGL1 goat IgG followed by Alexa Fluor® 546 labelled (red) donkey anti-goat IgG. Nuclei (blue) were stained with 4′,6′-diamidino-2-phenylindole dilactate. Images shown are representative of 3 independent experiments with 3–4 mice each.

Figure 2

MGL1^−/− mice exhibit reduced survival despite similar bacterial burden as the WT mice. (A) WT and MGL1^−/− mice were intranasally infected with 3.0x10⁴ CFUs of KPn in 20μl of sterile PBS and were assessed daily for disease severity. The survival was monitored for two week. Statistical comparison of susceptibility was done by Kaplan-Meier survival curve statistical analysis (p=0.0019 **). The data shown is from 3 independent experiments (n=16). (B) WT and MGL1^−/− were intranasally infected with KPn. At indicated times post infection the mice were sacrificed, systemic organs were isolated, homogenized and plated as described in Materials and Methods. Bacterial burden was enumerated after incubating the plates overnight at 37°C. No significant differences in bacterial burden (using non-parametric Mann-Whitney test) in WT and MGL1^−/− were found. Each symbol in the scatter plots represents individual mouse and the data is from 3–4 independent experiments.

Figure 3

MGL1 deficiency does not impair bacterial clearance by phagocytes. Bacterial uptake and killing capacity of MGL1^−/− and WT neutrophils (A) or bone-marrow derived macrophages (B) was determined at 1h and 3h by assessing intracellular CFUs in these cells as described in Methods. Data from a representative of 3 independent experiments is shown. (C) Neutrophils isolated from bronchoalveolar lavage fluid (BAL) of WT (upper panel) and MGL1^−/− (lower panel) mice infected with KPn, were cytocentrifuged and stained with Sytox Green to visualize extracellular traps (NETs). Magnification 200X. Bar graph shows quantitation of NET forming neutrophils in BAL from KPn infected WT and MGL1^−/− mice. Data are mean ± SEM from 8 mice per group in 3 independent experiments.

Figure 4

Pneumonic MGL1^−/− mice exhibit severe lung pathology and hyperinflammatory response. (A) Hematoxylin & Eosin staining of lung cryosections from mock control (i and v) and KPn infected WT (ii, iii, iv) and MGL1^−/− (vi, vii, viii) mice isolated at indicated times post-infection. Magnification 200X. (B) The lungs from mock control (WT-M and MGL1^−/− M) and KPn infected WT and MGL1^−/− (WT-Inf and MGL1^−/− Inf) mice isolated at 3d p.i. were homogenized in PBS with protease inhibitors and analyzed commercially for host immune mediators by rodent multi-analyte profile (Myriad^™ Rules-Based Medicine, Austin, TX). Results shown are mean ± SEM of 3–4 each infected and mock control mice from 2–3 independent experiments. Statistical significance are denoted by asterisks (*, p<0.05; **, p<0.005). CRP; C-reactive protein.

Figure 5

Pneumonic MGL1/^−/− mice exhibit increased neutrophil accumulation in their lungs. (A) Flow cytometry analysis of total lung cells from mock and KPn infected WT and MGL1^−/− at 3dp.i. Total lungs cells were isolated from mice by collagenase treatment followed by staining with antibodies against indicated cell markers as described in methods. Each bar represents percent cells positive for individual markers in lungs of indicated experimental mice. (B) Shows total number of cells positive for indicated cell markers. Data shown are the mean ± SEM of 5–6 mice from 3–4 independent experiments. Statistical significance are denoted by asterisks (*, p<0.05). (C) Ly6G+CD11b+ neutrophils in lungs of KPn infected WT and MGL1^−/− mice at 3dp.i. The cells were double-stained with anti-Ly6G-APC and anti-CD11b-Pacific Blue antibodies as markers for neutrophils. The bar graph shows mean ± SEM of total number of neutrophils in lungs of 3–4 mock control and 4–5 KPn infected WT and MGL1^−/− mice each from 3 independent experiments. Contour plots shown on the right are from one representative experiment. Statistical significance are denoted by asterisks (*, p<0.05).

Figure 6

Pneumonic MGL1/^−/− mice exhibit increased levels of neutrophil associated immune mediators. The lungs from mock control and KPn infected WT and MGL1^−/− mice were harvested at 3dp.i. and analyzed commercially for host immune mediators by rodent multi-analyte profile (Myriad^™ Rules-Based Medicine, Austin, TX). Levels of neutrophil chemoattractants and growth factor (CXCL1, CXCL6, GM-CSF) and activation markers (matrix metalloproteinase-9, MMP-9 and myeloperoxidase, MPO) are shown. Results shown are mean ± SEM of 3–4 each infected and mock control mice from 2–3 independent experiments. Statistical significance are denoted by asterisks (*, p<0.05; **, p<0.005).

Figure 7

MGL1 deficiency causes increased neutrophil influx in the pneumonic lungs. (A) Schematic representation of the adoptive transfer experiment as described in the Methods. Purified bone marrow neutrophils from WT mice were labelled with CellTracker Orange CMTMR and those from MGL1−/− mice labeled with CellTracker Green CMFDA, mixed in 1:1 ratio and injected intravenously (i.v.) into WT or MGL1^−/− recipient mice infected intranasally with KPn 24h prior to injection. Lungs were harvested 2 hrs after the adoptive transfer and processed for flow cytometry. (B) Relative recruitment of MGL1−/− and WT neutrophils in each mouse strain is depicted as the ratio of indicated populations in the KPn infected lungs. Data shown are mean ± SEM form 9 mice per group in 3 independent. Statistical significance are denoted by asterisks (**, p<0.005).