Innate Lymphocyte/Ly6C(hi) Monocyte Crosstalk Promotes Klebsiella Pneumoniae Clearance

Abstract

Increasing antibiotic resistance among bacterial pathogens has rendered some infections untreatable with available antibiotics. Klebsiella pneumoniae, a bacterial pathogen that has acquired high-level antibiotic resistance, is a common cause of pulmonary infections. Optimal clearance of K. pneumoniae from the host lung requires TNF and IL-17A. Herein, we demonstrate that inflammatory monocytes are rapidly recruited to the lungs of K. pneumoniae-infected mice and produce TNF, which markedly increases the frequency of IL-17-producing innate lymphoid cells. While pulmonary clearance of K. pneumoniae is preserved in neutrophil-depleted mice, monocyte depletion or TNF deficiency impairs IL-17A-dependent resolution of pneumonia. Monocyte-mediated bacterial uptake and killing is enhanced by ILC production of IL-17A, indicating that innate lymphocytes engage in a positive-feedback loop with monocytes that promotes clearance of pneumonia. Innate immune defense against a highly antibiotic-resistant bacterial pathogen depends on crosstalk between inflammatory monocytes and innate lymphocytes that is mediated by TNF and IL-17A.

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 day 5 post infection; (B) The survival rates of antibody treated (αLy6G or αGr1) WT and DT-treated CCR2-DTR mice. n=10; (C) colony forming units (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.

Figure 2. Adoptively transferred IMs promote bacterial clearance

(A) Schematic of experimental design; CD45.1⁺ WT or 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⁺CD11b^neg DCs, CD11b⁺CD103^neg cells and CD11b^negCD103^neg macrophages were further plotted for donor-derived CD45.2⁺ cells; (C) quantification of CD11c⁺CD11b⁺CD103^neg cell numbers shown in (B). n=3; (D) overlay of flow cytometry plots of donor-derived IMs before (blue) and 24 hours 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.

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 one day after infection. Data are represented as mean ± SEM. See also Figure S3.

Figure 4. TNF potentiates IL-17 production

A) Survival rates of WT mice treated with either anti-IL-17A blocking antibody (αIL-17A) 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 were 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 hours after infection. The values were normalized to those before infection (0 hour). Data are represented as mean ± SEM.

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 day 5. (B–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.

Figure 6. ILC3-mediated defenses 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 6H. n=3. Data are represented as mean ± SEM. See also Figure S4 and S6.

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 naïve 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 hours 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 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.