Differentiated THP-1 cells were infected with pneumococci (D39) and stained with (A, C) acridine orange (AO) and (B, D) JC-1 at the designated times post infection. (A–B) Representative histograms from one infection and (C–D) graphs summarizing loss of lysosomal acidification (LLA) and inner mitochondrial transmembrane potential (ΔΨ_m) in three separate experiments are shown, * = p<0.05, ** = p<0.01, one way ANOVA with Dunnett's post-test vs. 0 h. (E) AO staining 16 h post-infection of mock infected (Spn −) or pneumococcal infected (Spn+) differentiated THP-1 cells in the presence (+) or absence (−) of zVADfmk (zVAD) or zFAfmk (zFA), n = 3. Spn+ without zVAD/zFA vs. Spn+ with zVAD, p = ns (not significant) (F) Mock (Spn−) or D39 (Spn+) infected cells were stained with BODIPY FL-Pepstatin A 16 h post-infection and either visualized by microscopy or analyzed by flow cytometry. The filled histogram is Spn−, black histogram is Spn+, grey is unstained (US). The images and flow histograms are representative of three independent experiments. Scale bar equal to 5 µm. Quantified flow results are shown below the histogram, n = 3 (G) A Western blot of cytosolic and membrane fractions from mock (Spn−) or D39 (Spn+) infected differentiated THP-1 cells at 16 h post-infection probed with anti–cathepsin D (CatD) and cathepsin B (CatB). Actin and LAMP-1 were used as loading controls. The blots are representative of three independent experiments. (H) AO staining of differentiated THP-1 cells 16 h after mock-infection (MI) or exposure to D39 or a pneumolysin-deficient strain of D39 (PLYSTOP), n = 4, *** p<0.001, one-way ANOVA with Tukey's post-test. In all cases, pooled data are expressed as mean +/− SEM.

(A) Differentiated THP-1 cells were stained with JC-1 16 h after mock-infection (Spn−) or exposure to D39 pneumococci (Spn+) in the presence (+) or absence (−) of pepstatin A or MPC6. Loss of fluorescence indicates loss of ΔΨ_m, n = 3–5, * = p<0.05, ** = p<0.01, two-way ANOVA. (B) A representative Western blot of the cytosolic fractions of Spn− or Spn+ cells, 16 h after infection, in cultures incubated with (+) or without (−) pepstatin A (PepA). The blot is representative of four independent infections. (C) Spn- or Spn+ differentiated THP-1 cells, incubated in the presence (+) or absence (−) of pepstatin A (PepA), were assayed for caspase activity by fluorimetry 16 h post-infection, n = 5, * = p<0.05. (D) Spn− or Spn+ differentiated THP-1 cells, incubated in the presence (+) or absence (−) of pepstatin A or MPC6, were fixed and analyzed for nuclear fragmentation after 20 h culture, n = 3–4, * = p<0.05, ** = p<0.01, two-way ANOVA. In all cases, pooled data are expressed as mean +/− SEM.

(A) A representative Western blot for Mcl-1 from wild-type (WT) and Cathepsin D knock-out (KO) BMDMs 16 h after mock- (Spn−) or D39 pneumococcal-infection (Spn+), in the presence (+) or absence (−) of pepstatin A. The blot is representative of four independent experiments. Densitometry was carried out and fold change of Mcl-1 relative to mock-infection was calculated, n = 4 * = p<0.05, two-way ANOVA. (B) Spn− or Spn+ differentiated THP-1 cells were cultured in the presence (+) or absence (−) of pepstatin A (PepA). At 16 h cells were lysed, ubiquitinated proteins captured, and Western blots carried out probing for total ubiquitinated proteins and for Mcl-1. Densitometry was carried out and the ratios of Mcl-1 relative to ubiquitin were calculated, n = 3, * = p<0.05, one-way ANOVA with Tukey's post-test. (C) Spn− or Spn+ differentiated THP-1 cells were lysed and probed for Hsp70, Mule and actin at the designated times post-infection. The blots are representative of three experiments and the results summarized by densitometry.

(A) BMDMs from (WT) or cathepsin D knock-out (KO) mice were exposed to D39 pneumococci for the indicated time periods. Cells were lysed and intracellular bacteria plated out at the designated times, n = 5 per group, * = p<0.05, two-way ANOVA. (B) A representative Western blot of alveolar macrophages obtained from bronchial alveolar (BAL) fluid from irradiated mice transplanted with bone marrow from cathepsin D WT or KO mice. (C) The percentage of apoptotic alveolar macrophages in BAL in mice after adoptive transfer of marrow from WT or KO mice, 24 h after infection with 10⁴ type 1 pneumococci, as assessed by nuclear morphology, n = 6–10. The number of surviving bacteria in BAL 14 h (D) and 24 h (E) after infection with 10⁴ type 1 pneumococci, n = 5–9 ** = p<0.01, *** = p<0.001, students t-test. In all cases, pooled data are expressed as mean +/− SEM.

(A) A Western blot of phagolysosomes prepared from differentiated THP-1 cells 14 h after mock infection (MI), or infection with S. pneumoniae strain D39 and isolated using a discontinuous sucrose gradient was probed for cathepsin D. The blot is representative of three independent infections. (B) Cathepsin D activity was measured in whole cell lysates from mock (Spn−) or D39 (Spn+) infected differentiated THP-1 cells at the designated time points. D39 infected cells showed elevated cathepsin D activity compared to mock infected cells from 8 h, n = 4, *** = p<0.001, two-way ANOVA (C) Cathepsin D activity measured in whole cell lysates at 14 h in mock-infected (MI) cells, or differentiated THP-1 cells infected with the designated Spn strains (D39 or the pneumolysin-deficient strain PLYSTOP), Staph. aureus or latex beads, n = 4, ns =  not significant * = p<0.05. ** = p<0.01, *** = p<0.001 one-way ANOVA with Tukey's post-test. (D) Cytosolic pH was measured in mock (Spn−) or D39 (Spn+) infected cells at the designated time points using SNARF-4F carboxylic acid, acetoxymethyl ester, acetate, n = 4, * = p<0.05. ** = p<0.01, two-way ANOVA. (E) Cytosolic pH was measured at 14 h in differentiated THP-1 cells either mock-infected (Spn−) or exposed to D39 pneumococci (Spn+) in the presence (+) or absence (−) of pepstatin A (Pep A), n = 4, * = p<0.05, one-way ANOVA with Dunnett's post-test vs. MI. In all cases, pooled data are expressed as mean +/− SEM.

(A) BMDM's from (WT) or cathepsin D knock-out (KO) mice were stained with JC-1, 16 h after mock-infection (Spn−) or pneumococcal infection (Spn+) with D39 in the presence (+) or absence (−) of pepstatin A (PepA), n = 7, * = p<0.05, two-way ANOVA. (B) Nuclear fragmentation was detected by DAPI staining, in WT and cathepsin D KO BMDMs, 20 h after mock (Spn−) or D39 pneumococcal (Spn+) infection in the presence (+) or absence (−) of PepA, n = 5 per group. (C) Acridine orange staining of BMDMs 16 h post-infection in the presence (+) or absence (−) of pepstatin A, n = 7 per group, * = p<0.05, two-way ANOVA. In all cases, pooled data are expressed as mean +/− SEM.

(A) Mock-infected (Spn−) or D39 exposed (Spn+) differentiated THP-1 cells were immunoprecipitated with an anti-Mcl-1 antibody at the designated time period. As controls a separate sample at each time point was treated with a Mcl-1 peptide. The peptide used was identical to that used to produce the anti-Mcl-1 antibody. This excluded non-specific binding by the anti-Mcl-1 antibody. Precipitated proteins were blotted for Hsp70, Mule and Mcl-1. Densitometry was carried out and the ratios of Mule and Hsp70 to the amount of Mcl-1 precipitated was calculated, n = 3 * = p<0.05 for comparison of 4 h vs. 16 h, 1-way ANOVA with Dunnett's post test. Spn− or Spn+ differentiated THP-1 cells were cultured in the presence (+) or absence (−) of pepstatin A (PepA) for 16 h and lysates were immunoprecipitated with anti-Mcl-1 (B) or anti-Mule (C) antibody before being probed for Mule, Hsp70 and Mcl-1. C represents a control in which mock-infected (MI) cells were immunoprecipitated in the presence of an excess of antigen-specific peptide (Mcl-1) or a non-specific antibody for the immunoprecipitation of Mule. Densitometry was carried out and the ratios of the immunoblotted proteins to the immunoprecipitated Mcl-1 or Mule were calculated, n = 3 *** = p<0.001 for comparison of Spn+ with or without pepstatin A, one-way ANOVA with Tukey's post-test.

Mice were transplanted with bone marrow from cathepsin D deficient (KO) mice or with bone marrow from wild-type littermates (WT). Mice were instilled with (A) 10³ colony forming units of type 1 pneumococci for 24 h or (B) with 10⁴ type 1 pneumococci for 14 h and the number of neutrophils in BAL was calculated by analysis of cytospins. In all experiments, n = 3–9 * = p<0.05, students t-test. (C) Mice received bone marrow transplantation as above and WT or KO mice were instilled with 10⁴ type 1 pneumococci in the presence (+) of anti-Ly6G antibody or control antibody (−) to deplete neutrophils, n = 6 per group. In all cases, pooled data are expressed as mean +/− SEM.