Inflammatory (Ly6Chi CCR2+) monocytes provide defense against infections but also contribute to autoimmune diseases and atherosclerosis. Monocytes originate from bone marrow and their entry into the bloodstream requires stimulation of CCR2 chemokine receptor by monocyte chemotactic protein-1 (MCP1). How monocyte emigration from bone marrow is triggered by remote infections remains unclear. We demonstrated that low concentrations of Toll-like receptor (TLR) ligands in the bloodstream drive CCR2-dependent emigration of monocytes from bone marrow. Bone marrow mesenchymal stem cells (MSCs) and their progeny, including CXC chemokine ligand (CXCL)12-abundant reticular (CAR) cells, rapidly expressed MCP1 in response to circulating TLR ligands or bacterial infection and induced monocyte trafficking into the bloodstream. Targeted deletion of MCP1 from MSCs impaired monocyte emigration from bone marrow. Our findings suggest that bone marrow MSCs and CAR cells respond to circulating microbial molecules and regulate bloodstream monocyte frequencies by secreting MCP1 in proximity to bone marrow vascular sinuses.

Microbial penetration of the intestinal epithelial barrier triggers inflammatory responses that include induction of the bactericidal C-type lectin RegIIIγ. Systemic administration of flagellin, a bacterial protein that stimulates Toll-like receptor 5 (TLR5), induces epithelial expression of RegIIIγ and protects mice from intestinal colonization with antibiotic-resistant bacteria. Flagellin-induced RegIIIγ expression is IL-22-dependent, but how TLR signaling leads to IL-22 expression is incompletely defined. Using conditional depletion of lamina propria dendritic cell (LPDC) subsets, we demonstrated that CD103+ CD11b+ LPDCs, but not monocyte-derived CD103− CD11b+ LPDCs, expressed high amounts of IL-23 following bacterial flagellin administration and drove IL-22-dependent RegIIIγ production. Maximal expression of IL-23 subunits IL-23p19 and IL-12p40 occurred within 60 minutes of exposure to flagellin. IL-23 subsequently induced a burst of IL-22 followed by sustained RegIIIγ expression. Thus, CD103+ CD11b+ LPDCs, in addition to promoting long-term tolerance to ingested antigens, also rapidly produce IL-23 in response to detection of flagellin in the lamina propria.

Differences between TLR-deficient mouse colonies occur from extended husbandry in isolation that are communicated to offspring by maternal transmission.

Summary Infection with the opportunistic enteric pathogen Clostridium difficile is an increasingly common clinical complication that follows antibiotic treatment-induced gut microbiota perturbation. Innate lymphoid cells (ILCs) are early responders to enteric pathogens; however, their role during C. difficile infection is undefined. To identify immune pathways that mediate recovery from C. difficile infection, we challenged C57BL/6, Rag1−/−, which lack T and B cells, and Rag2−/− Il2rg−/− (Ragγc−/−) mice, which additionally lack ILCs, with C. difficile. In contrast to Rag1−/− mice, ILC-deficient Ragγc−/− mice rapidly succumbed to infection. Rag1−/−, but not Ragγc−/− mice, upregulate expression of ILC1 or ILC3 associated proteins following C. difficile infection. Protection against infection was restored by transferring ILCs into Ragγc−/− mice. While ILC3s made a minor contribution to resistance, loss of IFN-γ or T-bet-expressing ILC1s in Rag1−/− mice increased susceptibility to C. difficile. These data demonstrate a critical role for ILC1s in defense against C. difficile.

Chemokine receptor-mediated recruitment of inflammatory cells is essential for innate immune defense against microbial infection. Recruitment of Ly6Chigh inflammatory monocytes from bone marrow to sites of microbial infection is dependent on CCR2, a chemokine receptor that responds to MCP-1 and MCP-3. Although CCR2−/− mice are markedly more susceptible to Listeria monocytogenes infection than are wild-type mice, MCP-1−/− mice have an intermediate phenotype, suggesting that other CCR2 ligands contribute to antimicrobial defense. Herein, we show that L. monocytogenes infection rapidly induces MCP-3 in tissue culture macrophages and in serum, spleen, liver, and kidney following in vivo infection. Only cytosol invasive L. monocytogenes induce MCP-3, suggesting that cytosolic innate immune detection mechanisms trigger chemokine production. MCP-3−/− mice clear bacteria less effectively from the spleen than do wild-type mice, a defect that correlates with diminished inflammatory monocyte recruitment. MCP-3−/− mice have significantly fewer Ly6Chigh monocytes in the spleen and bloodstream, and increased monocyte numbers in bone marrow. MCP-3−/− mice, like MCP-1−/− mice, have fewer TNF- and inducible NO synthase-producing dendritic cells (Tip-DCs) in the spleen following L. monocytogenes infection. Our data demonstrate that MCP-3 and MCP-1 provide parallel contributions to CCR2-mediated inflammatory monocyte recruitment and that both chemokines are required for optimal innate immune defense against L. monocytogenes infection.

SUMMARY Antibiotic-mediated microbiota destruction and the consequent loss of colonization resistance can result in intestinal domination with vancomycin-resistant Enterococcus (VRE), leading to bloodstream infection in hospitalized patients. Clearance of VRE remains a challenging goal that, if achieved, would reduce systemic VRE infections and patient-to-patient transmission. Although obligate anaerobic commensal bacteria have been associated with colonization resistance to VRE, the specific bacterial species involved remain undefined. Herein we demonstrate that a precisely defined consortium of commensal bacteria containing the Clostridium cluster XIVa species Blautia producta and Clostridium bolteae restores colonization resistance against VRE and clears VRE from the intestines of mice. While C. bolteae did not directly mediate VRE clearance it enabled intestinal colonization with B. producta, which directly inhibited VRE growth. These findings suggest that therapeutic or prophylactic administration of defined bacterial consortia to individuals with compromised microbiota composition may reduce inter-patient transmission and intra-patient dissemination of highly antibiotic-resistant pathogens.

Aspergillus fumigatus is an environmental fungus that causes invasive aspergillosis (IA) in immunocompromised patients. Although -CC-chemokine receptor-2 (CCR2) and Ly6C-expressing inflammatory monocytes (CCR2+Mo) and their derivatives initiate adaptive pulmonary immune responses, their role in coordinating innate immune responses in the lung remain poorly defined. Using conditional and antibody-mediated cell ablation strategies, we found that CCR2+Mo and monocyte-derived dendritic cells (Mo-DCs) are essential for innate defense against inhaled conidia. By harnessing fluorescent Aspergillus reporter (FLARE) conidia that report fungal cell association and viability in vivo, we identify two mechanisms by which CCR2+Mo and Mo-DCs exert innate antifungal activity. First, CCR2+Mo and Mo-DCs condition the lung inflammatory milieu to augment neutrophil conidiacidal activity. Second, conidial uptake by CCR2+Mo temporally coincided with their differentiation into Mo-DCs, a process that resulted in direct conidial killing. Our findings illustrate both indirect and direct functions for CCR2+Mo and their derivatives in innate antifungal immunity in the lung.

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.