SUMMARY 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.

Delayed T-cell recovery and restricted T-cell receptor (TCR) diversity after allogeneic hematopoietic stem cell transplantation (allo-HSCT) are associated with increased risks of infection and cancer relapse. Technical challenges have limited faithful measurement of TCR diversity following allo-HSCT. Here we combined 5′-RACE PCR with deep sequencing, to quantify TCR diversity in 28 allo-HSCT recipients using a single oligonucleotide pair. Analysis of duplicate blood samples confirmed that the frequency of individual TCRs was accurately determined. After 6 months, cord blood graft recipients approximated the TCR diversity of healthy individuals, whereas recipients of T-cell-depleted peripheral blood stem cell grafts had a 28-fold and 14-fold lower CD4+ and CD8+ T-cell diversity, respectively. After 12 months, these deficiencies had improved for the CD4+, but not the CD8+ T-cell compartment. Overall, this method provides unprecedented views of T-cell repertoire recovery after allo-HSCT and may identify patients at high risk of infection or relapse.

Inflammatory monocytes (Ly6Chi CCR2hi) play a critical role in steering the course of responses against microbial infections of the lung. Lung monocytes can take up live fungi or bacteria and transport them to draining mediastinal lymph nodes (mLN). How microbes are taken up and trafficked by monocytes from the pulmonary site of infection is poorly understood. To study monocyte-mediated microbial trafficking, we developed a system to trace monocytes that carry particles from the lung to mLNs. Herein, we used mice expressing GFP or the diphtheria toxin receptor under the control of the CCR2 promoter to determine the impact of intratracheally instilled toll-like receptor (TLR) agonists on trafficking of with fluorescent, polystyrene microspheres from the lung to mLNs. This system recapitulates monocyte recruitment and trafficking seen during in vivo infections. Monocytes infiltrated the interstitial space in response to TLR2 agonists. And while neutrophils were the primary cell population associated with instilled microspheres in the lung, they did not deliver them independently to mLNs. Conversely, adoptively transferred monocytes successfully delivered microspheres to mLNs of CCR2+-cell-depleted mice. Our current system and findings empower us to further dissect the process of monocyte uptake of inhaled particles and antigen delivery to lymphocytes of mLNs, which may in turn inform novel vaccine strategies in an age of increasing emergence of antibiotic-resistant pathogens.

Molecules derived from gut-colonizing microbes contribute to intestinal homeostasis by stimulating innate immune receptors and maintaining a basal level of activation of host antimicrobial defenses. Depletion of intestinal microbes by treatment with antibiotics severely dampens these responses, increasing the risk for colonization and infection with pathogenic bacteria. A major consequence of antibiotic treatment is downregulation of the antimicrobial protein RegIII-γ. RegIII-γ expression is markedly decreased during antibiotic treatment but can be induced by administration of Toll-like receptor (TLR) ligands derived from Gram-negative bacteria. This observation suggests a role for Gram-negative organisms in stimulating RegIII-γ intestinal levels. In this study, we investigated the ability of several E. coli and K. pneumoniae strains to induce RegIII-γ in the mouse intestine during antibiotic stress. We find that although both bacterial species colonize the gastrointestinal tract of antibiotic-treated mice, only E. coli restores RegIII-γ expression in the ileum. E. coli, but not K. pneumoniae, induces IL-22 expression, a requisite step for RegIII-γ, induction, in the small intestinal lamina propria. Flagellin and TLR5 signaling are not required for E.coli-mediated RegIII-γ induction. Our findings suggest that Gram-negative bacteria colonizing the intestine differ in their ability to mediate IL-22-dependent RegIII-γ.