1. Volume 165, Issue 3, Pages 679-689 (April 2016)
Innate Lymphocyte/Ly6Chi Monocyte Crosstalk Promotes Klebsiella Pneumoniae Clearance 
Huizhong Xiong, James W. Keith, Dane W. Samilo, Rebecca A. Carter, Ingrid M. Leiner, Eric G. Pamer 
Cell 
Volume 165, Issue 3, Pages (April 2016)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1
Deficiency of K. pneumoniae Clearance in Monocyte-Depleted Mice
(A) Wild-type (WT) mice were inoculated with 2 × 105 CFUs of K. pneumoniae (Kp-MH258) intratracheally. The number of inflammatory monocytes (IMs) and neutrophils in the lung was measured from day 0 to 5 post-infection.
(B) The survival rates of antibody-treated (αLy6G or αGr1) WT and DT-treated CCR2-DTR mice. n = 10.
(C) CFUs of K. pneumoniae in the lungs on day 1 post-infection.
(D) Weight changes (left) and body temperature (on day 1 following infection, right). Data are represented as mean ± SEM.
See also Figure S1.
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4. Figure 2 Adoptively Transferred IMs Promote Bacterial Clearance
(A) Schematic of experimental design; CD45.1+ DT-treated CCR2-DTR mice were adoptively transferred with 6 × 106 CD45.2+ IMs from CCR2-GFP mice (+) or PBS (−) 30 min after K. pneumoniae (Kp) infection.
(B) Representative flow cytometry plots of CD11c+ cells in the lungs of the recipient mice. CD103+CD11bneg DCs, CD11b+CD103neg cells, and CD11bnegCD103neg macrophages were further plotted for donor-derived CD45.2+ cells.
(C) Quantification of CD11c+CD11b+CD103neg cell numbers shown in (B). n = 3.
(D) Overlay of flow cytometry plots of donor-derived IMs before (blue) and 24 hr after (red) transfer to infected recipients.
(E) CFUs of K. pneumoniae on day 1 post-infection in the lungs of WT and DT-treated CCR2-DTR mice with (+) or without (−) IM transfer. Data are represented as mean ± SEM.
See also Figure S2.
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5. Figure 3 TNF Production by Activated IMs
(A) Flow cytometry plots showing TNF+ cells in the whole-lung homogenates of WT and DT-treated CCR2-DTR mice on day 1 following infection (left); TNF+ cells were CD11c+ and were further plotted on the right for CD11b and CD103 expression.
(B) TNF+ cell numbers among different cell types. n = 3.
(C) CFUs in the lungs from WT, TNFR1−/−, and TNF−/− mice 1 day after infection. Data are represented as mean ± SEM.
See also Figure S3.
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6. Figure 4 TNF Potentiates IL-17 Production
(A) Survival rates of WT mice treated with either anti-IL-17A (αIL-17A) blocking antibody or isotype control. n = 4; the statistical analysis of survival rates was performed using Prism software.
(B) CFUs of the lungs from αIL-17A or isotype-treated WT mice, as well as from IL-17A−/− mice, on day 1 following infection.
(C) Flow cytometry of IL-17/IL-22 staining of infected lungs from WT, TNFR1−/−, and DT-treated CCR2-DTR mice (left); statistical analysis is shown on the right. n = 3.
(D) CFUs in infected lungs from WT, TNF−/−, and DT-treated CCR2-DTR animals administered with either αIL-17A or isotype control.
(E) Relative fold increase of IM numbers, TNF mRNA level and IL-17A+ cell numbers in the infected lungs of WT animals during the first 3 hr after infection. The values were normalized to those before infection (0 hr). Data are represented as mean ± SEM.
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7. Figure 5 Innate Lymphoid Cells Protect against K. pneumoniae Infection
(A) WT and Rag2−/− mice were infected and the bacterial burden in the lung was determined from day 1 to 5.
(B and C) Lung CFUs on day 1 following infection (B) and survival rates (C, n = 6) of WT, Rag2−/−, and Rag2/cγc−/− mice.
(D) CFUs in the infected lungs of Rag2−/− mice treated with anti-CD90 depleting antibody (αCD90) or isotype control. Data are represented as mean ± SEM.
See also Figure S5.
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8. Figure 6 ILC3-Mediated Defenses Occur through IL-17
(A) CFUs in the lung of Rag2−/− mice treated with αIL-17A or isotype control on day 1 post-infection.
(B) Flow cytometry analysis of IL-17A+ cells in Rag2−/− mice.
(C) The bacterial burden (left) and weight change (right) of Rag2/cγc−/− animals that were administered with recombinant murine IL-17 (rmIL-17) or PBS.
(D) Survival rates of the animals shown in (C).
(E) Rag2/cγc−/− mice were crossed to CCR2-DTR mice to generate Rag2/cγc−/− CCR2-DTR mice. The animals were infected, and the CFUs in the lungs were determined on day 1 following infection.
(F) Frequency of ILCs in the lungs of Rag2−/− mice treated with αTNF, DT-treated Rag2−/− CCR2-DTR, and Rag2−/− mice following infection.
(G) mRNA level of CCL20 in the lungs of the mice in (F).
(H) Flow cytometry (left) and statistical analysis (right) of the ILC frequencies in DT-treated CCR2-DTR mice transferred with IMs or PBS as depicted in Figure 2.
(I) Representative RORγt and GATA3 staining of ILCs identified in (H). n = 3. Data are represented as mean ± SEM.
See also Figures S4 and S6.
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9. Figure 7 Enhanced Antimicrobial Activities of IMs by IL-17
(A) Mean fluorescence intensity (MFI) of IL-17RA expression on different cell types; the IMs included both naive and activated inflammatory monocytes.
(B) IMs and neutrophils were purified on day 1 post-infection using MACS beads, washed and plated for cell-associated CFUs. The percentage of the cell-associated CFUs among the total CFUs was calculated. n = 3.
(C) The ratio of CFUs 2 hr after the transfer over the initial input (IM-associated CFUs before transfer). n = 3. Data are represented as mean ± SEM.
(D) Bacterial counts in the IM-K. pneumoniae co-culture at the indicated time points.
(E) Percentage of reactive oxygen species (ROS)+ cells among activated IMs (ROS+%) in the co-culture with or without rmIL-17.
(F) TNF mRNA level from the lungs of WT and IL-17A−/− mice on day 1 after infection. The mRNA abundance was normalized to β-actin, and the relative increase was calculated based on the uninfected state.
See also Figure S7.
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10. Figure S1
Deficiency of K. pneumoniae Clearance in Monocyte-Depleted Mice, Related to Figure 1
(A) CFUs of K. pneumoniae in the lungs (left), spleens (middle) and mediastinal lymph nodes (mLN, right) of WT and DT-treated CCR2-DTR animals from day 1 to day 4 following infection.
(B) Cell counts of neutrophils in BALF on day 1 post infection. n = 3.
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11. Figure S2
Activation of Adoptively Transferred IMs in Infected Hosts, Related to Figure 2
(A) The antibody panel used for fluorescence-activated cell sorting of IMs from CCR2-GFP donors (left) and purity check before/after sorting (right).
(B) CFUs of K. pneumoniae on day 3 post infection in the lungs of WT and DT-treated CCR2-DTR mice with (+) or without (-) a single dose of IM transfer.
(C) Representative flow cytometry plots of CD11c+ cells in the lungs of the recipient mice on day 1 (top) and day 3(bottom) following infection. The three major CD11c+ cell subsets were further plotted for donor-derived CD45.2+ cells.
(D) Flow cytometry plots of donor-derived CD45.2+ cells in different organs of the recipient mice. The cells were further plotted for Ly6C and CD11b.
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12. Figure S3 TNF Production by Activated IMs, Related to Figure 3
(A) Intracellular TNF staining of different cell subsets in uninfected (uninf) and infected (Kp-MH258) animals.
(B) WT animals were inoculated with 1.0 × 106 CFUs of K. pneumoniae. The TNF production by activated IMs was evaluated on day 1 following infection. In “TNF high” producers, greater than 20% of activated monocytes (CD11c+CD11b+CD103neg) cells were TNF-producers. The others, whose TNF-producing cell frequency was lower than 20%, were grouped as “TNF low” producers.
(C) CFUs in the lung of mixed bone marrow chimera of CCR2-DTR and TNF−/− (or WT or TNFR1−/−). Recipients were treated with DT prior to infection.
(D) CFUs from the lungs of WT and MyD88−/− mice on day 1 after infection.
(E) Tnf mRNA levels from the infected lung of WT and MyD88−/− mice.
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13. Figure S4
Characterization of IL-17A-Producing ILC3s, Related to Figure 6
(A) Representative flow cytometry plot of IL-17/IL-22 staining in the infected lungs of Rag2−/− mice. Cells were stimulated in vitro with ionomycin, PMA and IL-23. IL-17A+ cells were negative for both Lineage marker channels (see the Experimental Procedures), positive for CD45, CD90, RORγt. Overlay of IL-17A+ ILC3s (blue) and IL-17A− ILCs (predominantly ILC2s, red) demonstrated high expression of CCR6 and RORγt in ILC3s (right).
(B) Neutrophil stainings in the infected lungs of WT, αIL-17A-treated, and IL-17A−/− animals on day 1 following infection.
(C) IL-22−/− animals were inoculated with 2 × 105 CFUs of K. pneumoniae. CFUs in the lung (left) and weight changes (middle) were recorded on day 1 following infection. CFUs in different organs on day 3 post infection were shown on the right.
(D) Lung CFUs of Rag2/cγc−/− mice receiving transfer of Lin−CD45+CD90+ ILCs from Rag2−/− donors, IL-17A/Rag2−/− donors, or PBS.
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14. Figure S5 αCD90-Mediated ILC Depletion, Related to Figure 5
(A and B) Weight changes (A) and survival rates (B) of Rag2−/− mice treated with aCD90 or isotype control.
(C) IL-17A−/− and CCR2-DTR mice (treated with DT) were inoculated i.n. with 1x106 CFUs of K.pneumoniae strain Kp-MH258 or Kp-MH1867. Bacterial loads from the lung were measured on day 1 after infection.
(D) Il-17 mRNA level was measured from (C). The mRNA abundance was normalized to β-actin and the relative increase was calculated based on the uninfected state.
(E) staining and characterization of IL-17A+ cells from Rag2−/− IL17-GFP mice on day 1 after infection with Kp-MH258 or Kp-MH1867.
(F) isotype control or αCD90 depletion antibody was administered to Rag2−/− animals before K. pneumoniae inoculation. Bacterial burden from the lung was measured on day 1 after infection.
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15. Figure S6
IL-17 Production and Proliferation of ILCs, Related to Figure 6
(A) IL-17A staining of different immune cell types in the lungs of uninfected and infected (Kp-MH258) WT and Rag2−/− mice.
(B) BrdU staining on ILCs in the lungs of uninfected and infected Rag2−/− animals.
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16. Figure S7
Schematic of the Bacteria-Associated IM Transfer Experiment, Related to Figure 7
(A) Schematic of the experimental design of Figure 7C to study the impact of IL-17A on the bactericidal function of IMs. K. pneumoniae infected IMs (“IMs/Kp”) were purified as depicted in Figure 7B from infected WT or IL-17A−/− mice, and were adoptively transferred to naive WT animals intratracheally. Anti-IL-17A neutralizing antibody (αIL-17A) was administered to recipients of IL-17A−/− IMs to ensure IL-17A blockade after transfer (top). Free live K. pneumoniae (“free Kp”) and αIL-17A were inoculated intratracheally to WT as a control to monitor the effect of the αIL-17A antibody on the bacteria itself (bottom).
(B) Purity check of MACS-beads sorted infected cells. WT mice were inoculated with K. pneumoniae and the lung was harvested and homogenized on day 1 following infection. Neutrophils and IMs were purified sequentially according to the Experimental Procedures. Post-sorted cells (blue) were quantified, analyzed with flow cytometry, and compared with pre-sort cells (red). Note that this method enabled pooling of naive and activated IMs that are both CD11b+Ly6G− but differ in Ly6C expression (see also Figures 2D and S2D).
(C) PBS or 100ng/ml TNF was added to the IM-K. pneumoniae co-cultures and the bacterial loads were measured at indicated time points.
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