Journal: Microbiome

Article Title: Whole microbial community viability is not quantitatively reflected by propidium monoazide sequencing approach

doi: 10.1186/s40168-020-00961-3

Figure Lengend Snippet: PMA-seq identified viable microbes in simple synthetic communities. a Expected community structures of our ten live/dead Escherichia coli and Streptococcus sanguinis mixtures before and after PMA treatment: group (1) 100% live E. coli; 100% E. coli after PMA treatment; (2) 100% dead E. coli ; 0% E. coli after PMA treatment; (3) 100% live S. sanguinis ; 100% S. sanguinis after PMA treatment; (4) 100% dead S. sanguinis ; 0% S. sanguinis after PMA treatment; (5) 50% live E. coli and 50% live S. sanguinis ; same proportion after PMA treatment; (6) 50% dead E. coli and 50% dead S. sanguinis; no nucleotides after PMA-treatment; (7) 50% live E. coli , 25% live S. sanguinis and 25% dead S. sanguinis ; 67% E. coli and 33% S. sanguinis after PMA treatment; (8) 25% live E. coli and 25% dead E. coli ; 50% live S. sanguinis ; 67% S. sanguinis and 33% E. coli after PMA treatment; (9) 50% live E. coli , 50% dead S. sanguinis ; 100% E. coli after PMA treatment; 10) 50% dead E. coli and 50% live S. sanguinis ; 100% S. sanguinis after PMA treatment. b DNA quantity (ng) (top) and 16S rRNA gene copy numbers (bottom) of the 10 synthetic cultures with and without PMA treatment. Error bars represented the standard deviations. c Relative abundances of synthetic community members by PMA-seq before and after PMA treatment. Each experiment was carried out in quadruplex

Article Snippet: We constructed ten synthetic microbial communities (part 1) comprising viable or heat-killed E. coli strain ATCC 47076 and S. sanguinis strain ATCC 10556.

Techniques:

Journal: Microbiome

Article Title: Whole microbial community viability is not quantitatively reflected by propidium monoazide sequencing approach

doi: 10.1186/s40168-020-00961-3

Figure Lengend Snippet: PMA-treatment resulted in different degrees of compositional changes in different complex communities. Our second evaluation of PMA-seq used four environmental microbial community types (high and low biomass, high and low expected viability) spiked with varying levels of cultured / heat-killed E. coli . a Relative abundances of 15 taxa detected with the highest mean abundance across all samples. Each sample type contains four biological replicates. b Bray-Curtis distances within and between community samples with PMA treatment and samples without PMA treatment. Columns labeled with the sample type alone (e.g., screen) show dissimilarities within the indicated PMA-free samples. Those annotated “type(PMA)” (e.g., screen(PMA)) show calculations within the PMA-treated samples, and “type(between)” (e.g., screen(between) represents distances between paired samples with and without PMA treatment. c After constructing an ordination based on each sample pairwise Bray-Curtis dissimilarity, variation across the first explanatory axes is largely separated by human-associated uses, while the second axes appear to be explained by sample biomass. Here, lines connect identical samples with and without PMA treatment

Article Snippet: We constructed ten synthetic microbial communities (part 1) comprising viable or heat-killed E. coli strain ATCC 47076 and S. sanguinis strain ATCC 10556.

Techniques: Cell Culture, Labeling

Journal: Immunity

Article Title: Th17 cells mediate clade specific, serotype-independent mucosal immunity

doi: 10.1016/j.immuni.2011.10.018

Figure Lengend Snippet: Immunization with heat-killed K. pneumoniae induces antigen-specific Th17 responses. (A) Radial Cladogram of IL-17A(A), IL-17D, IL-17F protein families from different organisms including mammal (dark blue), bird (red), fish (sky blue), frog (pink), vase trunicate (lime) and oyster (green). Abbreviations: Hs: Homo Sapiens, Mm: Mus musculus, Bt: Bos Taurus, Rn: Rattus norvegicus, Ss: Sus scrofa, Ec: Equus caballus, Md: Monodelphis domestica, Ol: Oryzias latipes, Sr: Salmo salar, Tr: Takifugu rubripes, Dr: Danio rerio, Gg: Gallus gallus, Ci: Ciona intestinalis, Xt: Xenopus tropicalis, Cg: Crassostrea gigas (B) C57BL/6 mice were immunized intranasally with 20 µg heat-killed K. Pneumoniae-43816 (ATCC) (serotype K2) at Day 0 and Day 7 and sacrificed on Day14. Naïve mice were sacrificed 24h after 104 live K. Pneumoniae-43816 (ATCC) (serotype K2) infection. IL-17 and IL-22 producing cells in the lungs were analyzed by intracellular cytokine staining (upper panel). Subsets of IL-17 producing cells were determined by analyzing CD4 and γδ-TCR on IL-17 gated cells (lower panel). (C) Frequencies (left panel) and total numbers (right panel) of CD4 gated IFN-γ, IL-17A and IL-22 producing cells. (D) IL-17F-Th1.1 reporter mice were treated the same as in (B) and mononuclear cells in the lung were analyzed by flow cytometry. flow cytometry sorted splenic CD4+CD44lo (naïve CD4), splenic CD3+GL3+ (spleen GD), nTh-non17F, GD17 and Th17 were lysed in TriZol and RNA were extracted. Transcripts of cytokines were measured by real-time RT-PCR. Data represents two independent experiments. flow cytometry sorted Th17 from reporter mice were cultured with heat-killed K. pneumoniae, E. coli and S. aures in the presence of congenic CD45.1 splenocytes as APC for 4 days and responding Th17 cells were analyzed by staining congenic marker CD45.2 and CD4 (F). IL-17A released into the medium was measured by Luminex (G left panel) and total numbers were graphed (G right panel).

Article Snippet: To further support this, equal numbers of sorted GD17 and Th17 cell were cultured with heat-killed K. pneumoniae -43816 (ATCC), Th17 cells, but not GD17 cells, cells responded to K. pneumoniae- 43816 (ATCC) restimulation and produced IL-17 in the presence or absence of neutralizing antibodies against IL-1β and IL-23 ( Figure S1F ), indicating the antigen-specific memory Th17 cell recall response occurred independently of IL-1β and IL-23. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 Immunization with heat-killed K. pneumoniae induces antigen-specific Th17 responses. (A) Radial Cladogram of IL-17A(A), IL-17D, IL-17F protein families from different organisms including mammal (dark blue), bird (red), fish (sky blue), frog (pink), vase trunicate (lime) and oyster (green).

Techniques: Infection, Staining, Flow Cytometry, Quantitative RT-PCR, Cell Culture, Marker, Luminex

Journal: Immunity

Article Title: Th17 cells mediate clade specific, serotype-independent mucosal immunity

doi: 10.1016/j.immuni.2011.10.018

Figure Lengend Snippet: Characterization of immunization induced T cell responses. C57BL/6 mice were immunized intranasally with 20 µg of heat-killed K. Pneumoniae-43816 (ATCC) (serotype K2) at Day 0 and sacrificed 1, 2 and 4 weeks after. Lung, spleen and mediastinal lymph nodes were collected and intracellular staining was performed. Each time point has a group of 3 mice. (A) Representative flow cytometry plots of IL-17A and IFN-γ staining on CD4 gated cells in the spleen. (B) Representative flow cytometry plots of T-bet (upper panel) and Rorγt (lower panel) staining on IL-17A+ (Th17 blue line) and IFN-γ+ (Th1 red line) from the spleen. Mean fluorescent intensity of T-bet or Rorγt is shown on each plot. (C) Total numbers of Th1 (upper panel) and Th17 (lower panel) in lung, spleen and mediastinal lymph nodes. (D) Total numbers of Th1 and Th17 in three organs together were graphed. *Denotes significant higher cell numbers compared to naïve group.

Article Snippet: To further support this, equal numbers of sorted GD17 and Th17 cell were cultured with heat-killed K. pneumoniae -43816 (ATCC), Th17 cells, but not GD17 cells, cells responded to K. pneumoniae- 43816 (ATCC) restimulation and produced IL-17 in the presence or absence of neutralizing antibodies against IL-1β and IL-23 ( Figure S1F ), indicating the antigen-specific memory Th17 cell recall response occurred independently of IL-1β and IL-23. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 Immunization with heat-killed K. pneumoniae induces antigen-specific Th17 responses. (A) Radial Cladogram of IL-17A(A), IL-17D, IL-17F protein families from different organisms including mammal (dark blue), bird (red), fish (sky blue), frog (pink), vase trunicate (lime) and oyster (green).

Techniques: Staining, Flow Cytometry

Journal: Immunity

Article Title: Th17 cells mediate clade specific, serotype-independent mucosal immunity

doi: 10.1016/j.immuni.2011.10.018

Figure Lengend Snippet: Mucosal immunity to an autologous bacterial challenge is mediated by B-cells and Th17 cells. (A) C57BL/6 mice were immunized intranasally with 20 µg of heat-killed K. Pneumoniae-43816 (ATCC) (serotype K2) at Day 0 and Day 7. 4 weeks after the 2nd immunization, immunized mice and naïve unimmunized mice were infected with 104 live K. Pneumoniae-43816 (ATCC) (serotype K2) and sacrificed 24h after. Lung burden and systemic dissemination were determined by lung (left panel) and spleen (right panel) CFUs. Each group has an N>20 from 4 independent experiments. (B) 4 week after 2nd immunization, immunized mice and naïve mice were infected with 104 live K. Pneumoniae-43816 (ATCC) (serotype K2) and sacrificed 24h after. A group of immunized mice also received i.p. rat IgG or 1A8 24 hours and 2 hours prior to infection. A group of immunized mice received neutralizing antibody against IL-17A and/or IL-22 i.t. immediately prior to infection. Bacterial burdens were determined by lung CFU (left panel) and spleen CFU (middle panel) and neutrophil recruitment was determined by flow cytometry on Bronchoalveolar lavage cells (right panel). Each group has an N=5. (C) Rag2−/−Il2rg−/− mice were immunized intranasally with 20 µg K. Pneumoniae-43816 (ATCC) (serotype K2) at Day 0 and Day 7. Four weeks after the 2nd immunization, immunized mice and naïve unimmunized mice were infected with 104 live K. Pneumoniae-43816 (ATCC) (serotype K2) and sacrificed 24h after. Lung burden and systemic dissemination were determined by lung (left panel) and spleen (right panel) CFUs. Each group has 4–5 mice. (D) C57BL/6 mice were immunized intranasally with 20 µg heat-killed K. Pneumoniae-43816 (ATCC) (serotype K2) at Day 0 and Day 7. Four weeks after the 2nd immunization, immunized mice and naïve unimmunized mice were infected with 104 live K. Pneumoniae-43816 (ATCC) (serotype K2) and sacrificed 24h after. Anti- K. Pneumoniae-43816 (ATCC) (serotype K2) IgG and IgA in the serum and lung homogenate from naïve uninfected, infected naïve unimmunized and immunized mice were measured by ELISA. (E) C57BL/6 mice were immunized intranasally with 20 µg heat-killed K. Pneumoniae-43816 (ATCC) (serotype K2) at Day 0 and Day 7 and sacrificed on Day14. flow cytometry sorted B220+ cells from immunized mice lung were adoptively transferred into Rag2−/− Il2rg−/− mice. Rag2−/−Il2rg−/− mice receiving PBS or B-cells from immunized mice were infected with 104 live K. Pneumoniae-43816 (ATCC) (serotype K2) and sacrificed 24h later. Lung burden and systemic dissemination were determined by lung (left panel) and spleen (right panel) CFUs. Each group has N=4 mice. (F) Ighm−/− mice were immunized intranasally with 20 µg heat-killed K. Pneumoniae-43816 (ATCC) (serotype K2) at Day 0 and Day 7. 4 weeks after the 2nd immunization, immunized Ighm−/− mice and naïve-unimmunized Ighm−/− mice were infected with 104 live K. Pneumoniae-43816 (ATCC) (serotype K2) and sacrificed 24h after. A group of immunized mice also received i.p. rat IgG or 1A8 24h and 2h prior to infection. A group of immunized mice received neutralizing antibody against IL-17A i.t. immediately prior to infection. Bacterial burden were determined by lung CFU (left panel) and spleen CFU (right panel). Data are from two independent experiments with 2–4 mice in each group.

Article Snippet: To further support this, equal numbers of sorted GD17 and Th17 cell were cultured with heat-killed K. pneumoniae -43816 (ATCC), Th17 cells, but not GD17 cells, cells responded to K. pneumoniae- 43816 (ATCC) restimulation and produced IL-17 in the presence or absence of neutralizing antibodies against IL-1β and IL-23 ( Figure S1F ), indicating the antigen-specific memory Th17 cell recall response occurred independently of IL-1β and IL-23. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 Immunization with heat-killed K. pneumoniae induces antigen-specific Th17 responses. (A) Radial Cladogram of IL-17A(A), IL-17D, IL-17F protein families from different organisms including mammal (dark blue), bird (red), fish (sky blue), frog (pink), vase trunicate (lime) and oyster (green).

Techniques: Infection, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Immunity

Article Title: Th17 cells mediate clade specific, serotype-independent mucosal immunity

doi: 10.1016/j.immuni.2011.10.018

Figure Lengend Snippet: Cross-serotype protection with heterologous bacterial challenge requires Th17 cells, not B-cells. (A) Various stains of heat-killed K. pneumoniae were coated on a ELISA plate and lung homogenate from naïve mice (labelled as N) and K. Pneumoniae-43816 (ATCC) (serotype K2) immunized mice (labelled as V) were tested for cross reactivity. K. Pneumoniae strain numbers and serotypes are also listed. (B) Mediastinal lymph nodes from immunized C57BL/6 mice were labelled with CFSE and cultured with different serotypes of heat-killed K. Pneumoniae, E. coli, S. aureus and S. pneumoniae for 3 days. Proliferation of IL-17+ cells was analyzed by intracellular IL-17 staining and CFSE dilution. C57BL/6 mice were immunized intranasally with 20 µg heat-killed K. Pneumoniae-43816 (ATCC) (serotype K2) at Day 0 and Day 7. 10 days after the 2nd immunization, immunized mice and naïve unimmunized mice were infected with 104 K. Pneumoniae-303 (serotype K16) (C), K. Pneumoniae-396 (serotype K1) (D) or K. Pneumoniae-NDM1+(serotype unknown) (E) and sacrificed 24h after. Lung burden and systemic dissemination were determined by lung (left panel) and spleen (right panel) CFUs. Each groups has 4–5 mice. (F)Ighm−/− mice were immunized intranasally with 20 µg heat-killed K. Pneumoniae-43816 (ATCC) (serotype K2) at Day 0 and Day 7. Four weeks after the 2nd immunization, immunized Ighm−/− mice and naïve unimmunized Ighm−/− mice were infected with 104 live K. pneumoanie-396 (serotype K1) and sacrificed 24h after. A group of immunized mice also received i.t. neutralizing antibody against IL-17A right before infection. Bacterial burden were determined by lung CFU (left panel) and spleen CFU (right panel). Th17 responses in the lung were analyzed by RT-PCR (G). Data are from two independent experiments with 3–4 mice each group. (H) IL-17F-Thy1.1 reporter mice were immunized with heat-killed K. Pneumoniae-43816 (ATCC) (serotype K2) and were sacrificed one week after. Lung single cells were prepared and stained with CD4 and Thy1.1. flow cytometry sorted 105 Th17 (CD4+Thy1.1+) per mouse or 105 B cells (CD19+CD4− Thy1.1−) per mouse were intravenously transferred to Rag2−/− mice. Four week after transfer, mice were infected with 104 K. pneumoanie-396 (serotype K1) and sacrificed at 24h. Lung burdens were assessed by CFU. Each group has an N=5.

Article Snippet: To further support this, equal numbers of sorted GD17 and Th17 cell were cultured with heat-killed K. pneumoniae -43816 (ATCC), Th17 cells, but not GD17 cells, cells responded to K. pneumoniae- 43816 (ATCC) restimulation and produced IL-17 in the presence or absence of neutralizing antibodies against IL-1β and IL-23 ( Figure S1F ), indicating the antigen-specific memory Th17 cell recall response occurred independently of IL-1β and IL-23. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 Immunization with heat-killed K. pneumoniae induces antigen-specific Th17 responses. (A) Radial Cladogram of IL-17A(A), IL-17D, IL-17F protein families from different organisms including mammal (dark blue), bird (red), fish (sky blue), frog (pink), vase trunicate (lime) and oyster (green).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Staining, Infection, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry

Journal: Immunity

Article Title: Th17 cells mediate clade specific, serotype-independent mucosal immunity

doi: 10.1016/j.immuni.2011.10.018

Figure Lengend Snippet: Cross-serotype protection in heterologous challenge is mediated by Th17 not Th1. (A) C57BL/6 and Ifng−/− mice were immunized intranasally with 20 µg heat-killed K. Pneumoniae-43816 (ATCC) (serotype K2) at Day 0 and Day 7. 28 days after the 2nd immunization, immunized mice and naïve-unimmunized mice were infected with 104 K. pneumoanie-396 (serotype K1) and sacrificed 24h after. Lung burden and systemic dissemination were determined by lung (left panel) and spleen (right panel) CFUs. Each group has 4–5 mice. (B) IL-17F-Thy1.1 reporter mice were immunized with heat-killed K. Pneumoniae-43816 (ATCC) (serotype K2) and were sacrificed one week after. Lung single cells were prepared and stained with CD4 and Thy1.1. flow cytometry sorted 105 Th17 (CD4+Thy1.1+) per mouse or 105 Th-non17F (CD4+Thy1.1−) per mouse were intravenously transferred to Rag2−/−Il2rg−/− mice. Four week after transfer, mice were infected with 104 K. Pneumoniae-396 (serotype K1) and sacrificed at 24h. Lung burdens were assessed by CFU. Each group has 3–4 mice. (C) IL-17F-Thy1.1 reporter mice were immunized with heat-killed K. Pneumoniae-43816 (ATCC) (serotype K2) and were sacrificed one week after. Lung single cells were prepared and stained with CD4 and Thy1.1. flow cytometry sorted 105 Th17 (CD4+Thy1.1+) per mouse were intravenously transferred to Rag2−/− mice. Four week after transfer, mice were infected with 104 K. Pneumoniae-396 (serotype K1). Control IgG, α-IL17RC or α-IFN-γ were given i.t. 12h after infection and mice were sacrificed at 24h after infection. Lung and spleen burdens were assessed by CFU. Each group has 4 mice. (D) Il17ra−/− mice with B-cell depletion (0.1mg anti-CD20 i.p. weekly) mice were immunized intranasally with 20µg heat-killed K. Pneumoniae-43816 (ATCC) (serotype K2) at Day 0 and challenge with 104 K. Pneumoniae-396 (serotype K1) on Day28. Control IgG or α-IFN-γ were given i.t. 12h after infection and mice were sacrificed at 24h after infection. Lung bacterial burdens were assessed by CFU. Each group has 4–5 mice.

Article Snippet: To further support this, equal numbers of sorted GD17 and Th17 cell were cultured with heat-killed K. pneumoniae -43816 (ATCC), Th17 cells, but not GD17 cells, cells responded to K. pneumoniae- 43816 (ATCC) restimulation and produced IL-17 in the presence or absence of neutralizing antibodies against IL-1β and IL-23 ( Figure S1F ), indicating the antigen-specific memory Th17 cell recall response occurred independently of IL-1β and IL-23. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 Immunization with heat-killed K. pneumoniae induces antigen-specific Th17 responses. (A) Radial Cladogram of IL-17A(A), IL-17D, IL-17F protein families from different organisms including mammal (dark blue), bird (red), fish (sky blue), frog (pink), vase trunicate (lime) and oyster (green).

Techniques: Infection, Staining, Flow Cytometry

Journal: Immunity

Article Title: Th17 cells mediate clade specific, serotype-independent mucosal immunity

doi: 10.1016/j.immuni.2011.10.018

Figure Lengend Snippet: The Th17 response to Klebsiella outer membrane proteins is partially MHC-II dependent. (A) flow cytometry sorted CD4 T cells from K. Pneumoniae-43816 (ATCC) (serotype K2) immunized mice mediastinal lymph nodes were labelled with CFSE and cultured with flow cytometry sorted CD11+ DCs from C57BL/6 or MHC-II KO spleen for 4 days in the presence or absence of 10µL/mL OMP from K. Pneumoniae-396 (serotype K1) or K. Pneumoniae-43816 (ATCC) (serotype K2). Th17 proliferation was measure by intracellular IL-17 staining and CFSE dilution. Data are representative from two independent experiments. (B) IL-1β levels in the culture supernatants were measured by Luminex. (C) C57BL/6 mice were immunized with heat-killed K. Pneumoniae-43816 (ATCC) (serotype K2) (labeled as HKKP) or 1µg LPS or 1µg LPS plus 10µg OMPs isolated from K. Pneumoniae-43816 (ATCC) (serotype K2). 7 days later, mice were sacrificed and lungs were harvested for intracellular IL-17 and IFN-γ analysis on gated CD4+ T cells by flow cytometry. (D) Total Th1 (CD4+IFN-γ+) and Th17 (CD4+IL-17+) cells were graphed. (E) 4 week after immunization, LPS or LPS+OMP immunized mice were challenged with 104 live K. Pneumoniae-396 (serotype K1), bacterial burden were determine by lung and spleen CFU. Data are from two independent experiments with 3–5 mice each group. (F) Ighm−/− mice were immunized with 10µg OMPs isolated from K. Pneumoniae-43816 (ATCC) (serotype K2) and challenged with K. Pneumoniae-396 (serotype K1) 4 weeks later. Control IgG or anti-IL17RC was given i.t.12h after infection. Mice were sacrificed at 24h and bacterial burden were determined by lung CFU. Each group has 5 mice.

Article Snippet: To further support this, equal numbers of sorted GD17 and Th17 cell were cultured with heat-killed K. pneumoniae -43816 (ATCC), Th17 cells, but not GD17 cells, cells responded to K. pneumoniae- 43816 (ATCC) restimulation and produced IL-17 in the presence or absence of neutralizing antibodies against IL-1β and IL-23 ( Figure S1F ), indicating the antigen-specific memory Th17 cell recall response occurred independently of IL-1β and IL-23. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 Immunization with heat-killed K. pneumoniae induces antigen-specific Th17 responses. (A) Radial Cladogram of IL-17A(A), IL-17D, IL-17F protein families from different organisms including mammal (dark blue), bird (red), fish (sky blue), frog (pink), vase trunicate (lime) and oyster (green).

Techniques: Flow Cytometry, Cell Culture, Staining, Luminex, Labeling, Isolation, Infection