1. Volume 12, Issue 1, Pages 34-46 (July 2012)
Chitinase 3-like-1 Promotes Streptococcus pneumoniae Killing and Augments Host Tolerance to Lung Antibacterial Responses 
Charles S. Dela Cruz, Wei Liu, Chuan Hua He, Adam Jacoby, Alex Gornitzky, Bing Ma, Richard Flavell, Chun Geun Lee, Jack A. Elias 
Cell Host & Microbe 
Volume 12, Issue 1, Pages (July 2012)
DOI: /j.chom
Copyright © 2012 Elsevier Inc. Terms and Conditions

2. Figure 1 Chi3l1 Is Induced During Streptococcus pneumoniae Infection
(A–C) WT, Chi3l1+/−, and Chi3l1−/− mice were infected with 105 cfu/mouse of Sp intratracheally or control (−). The levels of Chi3l1 protein were analyzed by ELISA in the bronchoalveolar lavage fluid (BAL) (A) and serum (B). Immunohistochemistry was used to localize Chi3l1 (C, closed arrows depict macrophages and open arrows, airway epithelial cells).
(D and E) Double-label immunohistochemistry with cell-specific antibodies (CD68, macrophages; CC10, airway epithelial cells). These are representative composites of five similar evaluations. Arrows depict double-labeled cells.
(F) Peritoneal macrophages were infected with Sp at 2.5 × 104 cfu/well and incubated for 0, 1, 6, or 12 hr. Chi3l1 was evaluated by ELISA.
(G) Peritoneal macrophages were infected with 0, 104, 2.5 × 104, or 105 cfu/well of bacteria, supernatants were collected 6 hr later, and Chi3l1 was measured by ELISA. The values in (A), (B), (F), and (G) are the mean (±SEM) of evaluations of a minimum of eight mice and are representative of three experiments. Scale bar, 50 μm. n.d., none detected. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p <
Cell Host & Microbe  , 34-46DOI: ( /j.chom )
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3. Figure 2
Chi3l1 Plays a Critical Role in the Control of Acute Pneumococcal Infection and Regulates Lung Inflammation and Injury
(A and B) WT or Chi3l1−/− mice were infected intratracheally with Sp, and body weight (expressed as % original body weight) (A) and mortality (B) were assessed.
(C–I) WT or Chi3l1−/− mice were infected intratracheally with Sp. Total BAL cell recovery and differential cell recoveries were assessed (C). Tissue histology (H&E stain) (D), RBC recovery (E), BAL protein (F), and the percentage and numbers of CD11b+Gr-1+ cells (G and H) were assessed via FACS analysis of single lung cell suspensions, as well as the levels of lung lysate myeloperoxidase (MPO) (I). Unless otherwise noted the data were obtained 4 days after infection. The values in (A), (C), (E), (F), (H), and (I) represent the mean (±SEM) of evaluations in a minimum of five animals and are representative of three experiments. NS, not significant. Scale bar, 100 μm. ∗p < 0.05, ∗∗p < (See also Figure S1.)
Cell Host & Microbe  , 34-46DOI: ( /j.chom )
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4. Figure 3 Effects of Recombinant Chi3l1
(A and B) WT and Chi3l1−/− mice were infected intratracheally with S. pneumoniae (Sp) or with Sp incubated with 10 μg/ml of rChi3l1. Daily weights of infected mice were recorded (A). Survival of WT or Chi3l1−/− mice infected with Sp or with Sp incubated with rChi3l1 was assessed (B). Black boxes, WT mice + Sp; white boxes, WT mice + Sp+rChi3l1; black triangles, Chi3l1−/− mice + Sp; white triangles, Chi3l1−/− mice + Sp+rChi3l1.
(C) Sp was incubated for 16 hr in the presence and absence of 5-fold dilutions of rChi3l1 (from 10 μg/ml to 16 ng/ml), doxycycline (1 μg/ml), or media control, and bacterial growth was assessed.
(D–F) WT and Chi3l1−/− mice were infected intratracheally with 5 × 105 cfu/mouse Sp. Lungs from infected WT and Chi3l1−/− mice were harvested 1 or 4 days postinfection, and tissue bacterial counts were determined (D). BAL from infected WT and Chi3l1−/− mice were prepared 4 days after inoculation, and bacterial counts were determined (E). Spleen lysates from WT and Chi3l1−/− mice were prepared 4 days after bacterial inoculation, and the percentage of mice with positive spleen cultures was assessed (F).
(G) WT and Chi3l1−/− mice were infected with 105 cfu Sp in the presence or absence of 10 μg/ml rChi3l1. One day later, lung lysate recovered bacterial load was determined by plating on blood agar.
(H) Peritoneal macrophages from WT or Chi3l1−/− mice were treated for 60 min with 10:1 and 2.5:1 ratios of fluorescein-labeled Sp to cells. Cells were gated on macrophages according to forward and side scatter properties. Average fluorescein units are shown for WT or Chi3l1−/− cells (n = 5).
(I) Peritoneal macrophages from WT or Chi3l1−/− mice were treated with a 10:1 ratio of unlabeled Sp to cells in media first without antibiotics for 60 min, followed by media with antibiotics for 60 min. The cells were then washed and lysed, and phagocytosed bacteria were quantitated (n = 4).
(J) Peritoneal macrophages from WT or Chi3l1−/− mice were treated with 10:1 ratio of unlabeled Sp to cells in media without antibiotics for 60 min, then incubated with gentamicin to kill extracellular bacteria, and viable bacteria were assessed 6 hr later (n = 4).
(K) Peritoneal macrophages from WT or Chi3l1−/− mice were treated with Sp in the absence or presence of rChi3l1, and at 6 hr, viable intracellular bacteria were assessed. The values in this figure represent the mean (±SEM) of evaluations in a minimum of five animals and are representative of three experiments. NS, not significant. ∗p < 0.05, ∗∗p < (See also Figure S2.)
Cell Host & Microbe  , 34-46DOI: ( /j.chom )
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5. Figure 4 Chi3l1 Regulates Macrophage Cell Death
(A) Peritoneal macrophages from WT or Chi3l1−/− mice were incubated with 0, 104, 2.5 × 104, and 105 cfu Sp/well for 12 hr. Cells were fixed and TUNEL stains were performed.
(B) Infected peritoneal macrophages were harvested and stained with Annexin V-FITC, and the percentage of cells that expressed Annexin V was determined.
(C) Macrophages from WT or Chi3l1−/− mice treated with Sp in the absence or presence of rChi3l1 and LDH release were assessed. The values are expressed as the percentage of the LDH released by similar numbers of cells treated with Triton-X.
(D) Macrophages from WT or Chi3l1−/− mice were treated with Sp in the presence or absence of rChi3l1, and the percentage of cells expressing Annexin V by flow cytometry was assessed.
(E) Peritoneal macrophages from WT or Chi3l1−/− mice were treated with Sp in the presence or absence of rChi3l1, and lysate caspase-1 bioactivity was measured using a colorimetric assay.
(F) CD45+ adherent cells from infected WT or Chi3l1−/− lungs were measured for their caspase-1 bioactivity.
(G) Annexin V staining by flow cytometry of macrophages (WT or Chi3l1−/−) infected with Sp in the presence of DMSO (vehicle control) or YVAD-CHO caspase-1 inhibitor.
(H) Annexin V staining by flow cytometry of macrophages from WT, Chi3l1−/−, Chi3l1−/− × Caspase-1−/−, or Caspase-1−/− mice infected with Sp. The data shown in (A)–(H) represent the mean ± SEM of a minimum of five mice in each assessment. NS, not significant. ∗p < (See also Figure S3.)
Cell Host & Microbe  , 34-46DOI: ( /j.chom )
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6. Figure 5
Chi3l1 Regulates Inflammasome Activation and the Purinergic Pathway
(A–C) WT and Chi3l1−/− mice were infected intratracheally with Sp, and BAL fluid IL-1β (A) and IL-18 (B) were assessed 1 or 4 days after inoculation. The levels of BAL TNF and IL-6 were assessed 4 days after inoculation (C). BAL fluids of mock infected animals were obtained at day 4.
(D–G) WT and Chi3l1−/− mice were infected with Sp and 1 or 4 days later the levels of mRNA encoding Nlrp3 (D) and ASC (E) in isolated lung lysates were assessed by real-time RT-PCR. Similarly, isolated macrophages from WT and Chi3l1−/− mice were infected in vitro with Sp and measured for Nlpr3 (F) and ASC (G) expression.
(H) WT and Chi3l1−/− mice were infected with Sp, and the levels of BAL ATP were assessed 4 days later.
(I and J) WT and Chi3l1−/− mice were infected with Sp, and the levels of mRNA encoding the purinergic receptors P2X7R (I) and P2X4R (J) were assessed using real-time RT-PCR. Expression is normalized to a β-actin control and presented as the fold increase compared to mock treated WT mice. Error bars represent mean ± SEM. NS, not significant. ∗p < 0.05, ∗∗p < (See also Figure S4.)
Cell Host & Microbe  , 34-46DOI: ( /j.chom )
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7. Figure 6
Chi3l1 Regulates an Inflammasome that Utilizes Caspase-1 and Nlpr3
(A and B) BAL cell numbers (A) and IL-1β levels in BAL (B) in Sp-infected WT mice, Chi3l1−/− mice, and mice treated with scrambled siRNA, Nlrp3 siRNA, and P2X7R siRNA.
(C) Peritoneal macrophages from WT or Chi3l1−/− mice were infected with Sp at 10:1 bacteria to cell ratio, and supernatant IL-1β was assessed by ELISA at the noted intervals thereafter.
(D) Macrophages from WT or Chi3l1−/− mice were treated with Sp as indicated for 16 hr, and lysates and supernatant (SN) IL-1β were assessed via western blot analysis. Pro-IL-1β and the mature form of IL-1β are indicated. A control immunoblot using antibodies against β-tubulin is included.
(E and F) Peritoneal macrophages from WT or Chi3l1−/− mice were treated with LPS for 12 hr followed by ATP (5 mM) for 30 min. The levels of supernatant IL-1β were assessed by ELISA (E), and supernatant and lysate IL-1β protein were assessed by immunoblot (F).
(G) Macrophages from WT or Chi3l1−/− mice treated with or without Sp, and 16 hr later caspase-1 was assessed via western blot analysis. Pro-caspase-1 and cleaved caspase-1 (p10) are shown. Control immunoblot using antibodies against β-tubulin is included.
(H) Macrophages from WT mice, Chi3l1−/− mice, caspase-1−/− mice, and Chi3l1−/− × Caspase-1−/− mice were infected with Sp, and supernatant IL-1β was assessed by ELISA 16 hr later.
(I) Macrophages from WT mice, Chi3l1−/− mice, Nlrp3−/− mice, and Chi3l1−/− × Nlrp3−/− mice were infected with Sp, and supernatant IL-1β was assessed by ELISA 16 hr later.
(J) BAL IL-1β levels in infected WT, Chi3l1−/− mice, and Chi3l1−/− × Caspase-1−/− mice. Error bars represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < (See also Figure S5.)
Cell Host & Microbe  , 34-46DOI: ( /j.chom )
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8. Figure 7 Chi3l1 Regulation of Cytokine Production In Vivo
(A–D) WT and Chi3l1−/− mice were infected with or without Sp. BAL was undertaken 4 days later, and Th1 cytokines (IL-12p70 and interferon-γ) (A), Th2 cytokines (IL-13, IL-4, and IL-5) (B), TSLP (C), and IL-17 (D) were measured using ELISA. The values in (A)–(D) represent the mean (±SEM) of evaluations in a minimum of eight animals and are representative of two experiments. NS, not significant. Scale bar, 100 um. ∗p < 0.05, ∗∗p < (See also Figure S6.)
Cell Host & Microbe  , 34-46DOI: ( /j.chom )
Copyright © 2012 Elsevier Inc. Terms and Conditions