Lack of Mincle or FcRγ does not influence the antibacterial host response during pneumococcal pneumonia.

<p>(A) C57Bl/6 mice were intranasally infected with 5×10⁶ CFU/mouse <i>S. pneumoniae</i> ST3 or control treated with PBS and Mincle expression levels in the lungs at the indicated time points were determined by quantitative RT-PCR. (B) WT, <i>Fcerg1^-/-</i> and <i>Mincle^-/-</i> mice were intranasally infected with 7.5×10⁴ CFU/mouse <i>S. pneumoniae</i> ST3 and survival was monitored every 12 h over 10 days. (C-K) WT, <i>Fcer1g^-/-</i> and <i>Mincle^-/-</i> mice were intranasally infected with 5×10⁶ CFU/mouse <i>S. pneumoniae</i> ST3 or treated with PBS. Bacterial loads were determined in the (C) bronchoalveolar lavage fluid (BALF) and (D) blood. (E) Neutrophils and (F) macrophages in the BALF were quantified by flow cytometry. (G) IL-6 and (H) TNFα (H) levels in the BALF were analyzed by ELISA. Relative expression of (I) <i>Cxcl1</i>, (J) <i>Ccl2</i> and (K) <i>Gcsf</i> in the lung was determined by quantitative RT-PCR. Data are shown as mean + SEM; (A) n = 3, (B) n = 8–10, (C-F) n = 6–22, (G-H) n = 6–8, (I-K) n = 4–5 mice each group. n.s. not significant.</p

Lack of FcRγ or Mincle does not affect the innate immune response to <i>S. pneumoniae</i> in different cell types.

<p>(A) AECs, MVECs and AMΦs were isolated from the lung, and PMNs and BMMs from the bone marrow of C57Bl/6 mice. Cells were left untreated or infected with <i>S. pneumoniae</i> ST2 (D39). (B-G) AMΦs, BMMs and PMNs were isolated from WT, <i>Fcer1g^-/-</i> and <i>Mincle^-/-</i> mice. (B, C) AMΦs or (D) BMMs were infected with <i>S. pneumoniae</i> ST2 or (E) BMMs were stimulated with TDM as a positive control for 16 h and cytokine release was quantified by ELISA. (F) Untreated or LPS-treated (100 ng/ml, 4 h) BMMs were infected with <i>S. pneumoniae</i> ST2Δ<i>cps</i> (MOI = 2.5) and were treated with gentamicin (50 mg/ml) after 30 min. Intracellular, viable bacteria were determined 60 min or 120 min post infection. (G) Rabbit serum-opsonized <i>S. pneumoniae</i> ST2Δ<i>cps</i> were incubated with PMNs and neutrophil-mediated killing was assessed after 45 min incubation. Data are shown as mean + SEM of (A) two (A: PMN, E, F), three (C, G), four (A: AEC, MVEC, B, D) or five (A: AMΦ, BMM) independent experiments carried out in duplicates (A-F) or quadruplicates (G); * = p<0.05; ** = p<0.01; *** = p<0.001; n.s. not significant.</p

The C-Type Lectin Receptor Mincle Binds to <i>Streptococcus pneumoniae</i> but Plays a Limited Role in the Anti-Pneumococcal Innate Immune Response

<div><p>The innate immune system employs C-type lectin receptors (CLRs) to recognize carbohydrate structures on pathogens and self-antigens. The Macrophage-inducible C-type lectin (Mincle) is a FcRγ-coupled CLR that was shown to bind to mycobacterial cord factor as well as certain fungal species. However, since CLR functions during bacterial infections have not yet been investigated thoroughly, we aimed to examine their function in <i>Streptococcus pneumonia</i> infection. Binding studies using a library of recombinantly expressed CLR-Fc fusion proteins indicated a specific, Ca²⁺-dependent, and serotype-specific binding of Mincle to <i>S. pneumonia</i>. Subsequent experiments with different Mincle-expressing cells as well as Mincle-deficient mice, however, revealed a limited role of this receptor in bacterial phagocytosis, neutrophil-mediated killing, cytokine production, and antibacterial immune response during pneumonia. Collectively, our results indicate that Mincle is able to recognize <i>S. pneumonia</i> but is not required for the anti-pneumococcal innate immune response.</p></div

Mincle binding to <i>S. pneumoniae</i> is Ca²⁺-dependent.

<p>(A) To analyze the Ca²⁺ dependency of the Mincle/<i>S. pneumoniae</i> interaction, TDB (50 µg/mL in isopropanol) and heat-inactivated <i>S. pneumoniae</i> ST3 (3×10⁸ cells/mL in PBS) were immobilized on ELISA plates. Binding of Mincle-Fc (10 µg/mL) diluted in lectin binding buffer or EDTA buffer (10 mM EDTA) to immobilized TDB and <i>S. pneumoniae</i> ST3 was analyzed. Data are representative of three independent experiments (triplicates each). (B) Mincle-Fc (20 µg/mL) (non-filled curves) or Fc only (gray-filled curves) were incubated with <i>S. pneumoniae</i> ST3 (3×10⁸ cells/mL) in lectin binding buffer or EDTA buffer (10 mM EDTA). Left: Representative histogram plot of one binding experiment. Right: Statistical analysis of the flow cytometry-based binding assay in the presence or absence of EDTA. Data are representative of three independent experiments (triplicates each). (C) 20 µg/mL Mincle-Fc, diluted in lectin binding buffer, was pre-incubated with <i>S. pneumoniae</i> ST3 and subsequently incubated with plate-bound TDB (50 µg/mL). Data are representative of three independent experiments (triplicates each). (A-C), Data are shown as mean + SEM. Significance is indicated by asterisks, * = p<0.05; ** = p<0.01; *** = p<0.001.</p

Mincle binds to <i>S. pneumoniae</i>.

<p>(A) Heat-inactivated <i>S. pneumoniae</i> ST3 (1.5×10⁷ cells/well) diluted in PBS were immobilized on ELISA plates and incubated with CLR-Fc fusion proteins (10 µg/mL) diluted in lectin binding buffer. Data are representative of three independent experiments (triplicates each). The dashed line indicates background binding of CLR-Fc fusion proteins. (B) Binding of Mincle-Fc, SIGNR1-Fc and DCIR-Fc was analyzed by flow cytometry. CLR-Fc fusion proteins (20 µg/mL) were incubated with <i>S. pneumoniae</i> ST3 at a concentration of 3×10⁸ cells/mL diluted in lectin binding buffer. Left: Representative histogram plot of one binding experiment. Right: Statistical analysis of the flow cytometry-based binding assay. Data are representative of three independent experiments (triplicates each). (C) Mincle-Fc binding to heat-killed <i>T. cutaneum</i> and <i>S. pneumoniae</i> ST3 was analyzed by flow cytometry. Percentage of binding is shown relative to Fc binding to each pathogen. Data are representative of three independent experiments (triplicates each). (A-C), Data are shown as mean + SEM. Significance is indicated by asterisks, * = p<0.05; ** = p<0.01; *** = p<0.001; **** = p<0.0001.</p

Rac1 controls IL-1β production at a posttranscriptional level in <i>C. pneumoniae</i>-infected cells.

<p>PBMCs were transfected with control siRNA or siRNA specific for Rac1. After 48 h, cells were infected with <i>C. pneumoniae</i> (MOI 3) for 16 hrs and knock down of Rac1 was assessed by reverse transcription PCR (A). Cell supernatants were subjected to IL-1β ELISA (B), and levels of pro-IL-1β mRNA were analyzed by Q-PCR (C). (D) THP-1 cells were incubated with the indicated concentrations of NSC23766 for 30 min and afterwards infected with <i>C. pneumoniae</i> (MOI 3) for 8 h. Cell lysates were assayed for pro-caspase-1 and caspase-1 p20 by Western blot. The western Blot is representative of three independent experiments. (E, F) THP-1 cells seeded on coverslips were treated or not treated with NSC23766, and infected with <i>C. pneumoniae</i> for 20 h. Bacteria (red) and ASC (green) were visualized by confocal laser scanning microscopy using specific antibodies. The arrowheads point to ASC foci. Images are representative of three independent experiments (original magnification 63×).</p

Role of Rac1 in the production of IL-1β in <i>C. pneumoniae</i>-infected cells.

<p>(A) PBMCs were incubated with different concentrations of the Rac1 inhibitor NSC23766 for 30 min and subsequently infected with <i>C. pneumoniae</i> (MOI 3), or (C) cells were first infected with <i>C. pneumoniae</i> (MOI 3) and NSC23766 was added 2.5 h post-infection. After incubating 16 hrs production of IL-1β was quantified by ELISA. Total RNA was harvested for quantification of chlamydial 16s rRNA production (B, D) using real-time PCR as indicated in the Materials and Methods section. (E) THP-1 cells were transfected with control siRNA or siRNA specific for Rac1. After 48 h, cells were infected with <i>C. pneumoniae</i> (MOI 3) for 16 hrs and knock down of Rac1 was assessed by reverse transcription PCR. (F) HEp-2 reinfection assay in which siRNA-transfected THP-1 cells infected with <i>C. pneumoniae</i> (MOI 0.5; 72 h) were harvested and inoculated onto monolayers of HEp-2 cells. Infected cells were then stained for Chlamydia 48 h p.i. and clamydial inclusions were counted. Data shown are representative for at least three (A–D) or two (E, F) experiments performed in duplicates.</p

<i>C. pneumoniae</i> stimulates production of mature IL-1β in human PBMCs.

<p>(A) Human PBMCs were infected with different MOI of <i>C. pneumoniae</i> for 16 hrs and production of IL-1β was determined by ELISA. (B) Human PBMCs were infected with <i>C. pneumoniae</i> (MOI 3) for different time intervals and amounts of mature IL-1β (17 kDA) in the cell supernatant was visualized by Western Blot. The western blot is representative of three independent experiments. Results obtained from ELISAs represent mean ± SD of three independent experiments.</p

Caspase-1, ASC and NLRP3 mediate IL-1β production in <i>C. pneumoniae</i>-infected cells.

<p>(A) THP-1 monocytes were infected with <i>C. pneumoniae</i> (MOI 3) for different time intervalls. Cell lysates were harvested and assayed for procaspase-1 and caspase-1 p20. (B, C) PBMCs were transfected with control siRNA or siRNA specific for caspase-1. After 48 h, cells were infected with <i>C. pneumoniae</i> (MOI 3) for 16 hrs. Expression of caspase-1 was examined by reverse transcription PCR, and supernatants were subjected to IL-1β ELISA. (D–G) PBMCs were transfected with control siRNA or siRNA specific for ASC (D, E) or NLRP3 (F, G). After 48 h, cells were infected with <i>C. pneumoniae</i> (MOI 3), expression of ASC (D) and NLRP3 (F) was examined by reverse transcription PCR, and supernatants were subjected to IL-1β ELISA (E, G). (H) Cells were transfected with siRNA as indicated and, after 48 h, infected with <i>C. pneumoniae</i> (MOI 3). Cell lysates were assayed for procaspase-1 and caspase-1 p20. (I) Mouse BMMs obtained from wildtype and Nlrp3−/− mice were infected with <i>C. pneumoniae</i> (MOI 3) for 16 hrs. Production of mIL-1β was quantified by ELISA. Western Blots are representative for at least three independent experiments. Results obtained from ELISAs represent mean ± SD of three independent experiments.</p

Type I IFN responses during <i>L</i>. <i>pneumophila</i> infection are mediated by the cGAS/STING pathway.

<p>(A-C) WT and <i>Tmem173</i>^-/- mouse BMDMs were left untreated or stimulated with 1 ug/ml <i>L</i>. <i>pneumophila</i> DNA (JR32 DNA) or 5 ug/ml 2´3-cGAMP (A) or were infected with <i>L</i>. <i>pneumophila</i> JR32 WT and 130b WT, or mutant strains deficient for <i>dotA</i> or <i>sdhA</i> at MOI 10 for 6 h (B, C). Expression of <i>Ifnb</i> (A, B) or <i>Irg1</i> (C) was measured by qRT-PCR. (D-G) WT and cGAS-deficient BMDMs were stimulated with <i>L</i>. <i>pneumophila</i> DNA or 2´3-cGAMP or infected with <i>L</i>. <i>pneumophila</i> JR32 WT, and expression of <i>Ifnb</i> and <i>Irg1</i> was quantified by qRT-PCR (D-F) or production of IP-10 was measured by ELISA (G). (H-N) WT, STING- and cGAS-deficient mice were intranasally infected with 1×10⁶ <i>L</i>. <i>pneumophila</i> JR32 WT or instilled with PBS as control (H-J). <i>Ifnb</i> and <i>Irg1</i> expression in the lungs was assessed 48 (H, I) or 144 h p.i. (K-N) by qRT-PCR, or IP-10 production was measured at 48 h (J). Data are represented as the relative quantification (RQ) of specified mRNAs. Data are shown as the mean + SEM of three to four independent experiments, measured in technical duplicates (Fig. 1A-G) or 6 to 7 mice per group (Fig. H-N). Analyses were performed through the Mann-Whitney U Test. Comparisons with a <i>p</i> < 0.05 were considered significant.</p