Irf4-dependent CD103+CD11b+ dendritic cells and the intestinal microbiome regulate monocyte and macrophage activation and intestinal peristalsis in postoperative ileus

Objective: Postoperative ileus (POI), the most frequent complication after intestinal surgery, depends on dendritic cells (DCs) and macrophages. Here, we have investigated the mechanism that activates these cells and the contribution of the intestinal microbiota for POI induction.Design: POI was induced by manipulating the intestine of mice, which selectively lack DCs, monocytes or macrophages. The disease severity in the small and large intestine was analysed by determining the distribution of orally applied fluorescein isothiocyanate-dextran and by measuring the excretion time of a retrogradely inserted glass ball. The impact of the microbiota on intestinal peristalsis was evaluated after oral antibiotic treatment.Results: We found that Cd11c-Cre+ Irf4flox/flox mice lack CD103+CD11b+ DCs, a DC subset unique to the intestine whose function is poorly understood. Their absence in the intestinal muscularis reduced pathogenic inducible nitric oxide synthase (iNOS) production by monocytes and macrophages and ameliorated POI. Pathogenic iNOS was produced in the jejunum by resident Ly6C- macrophages and infiltrating chemokine receptor 2-dependent Ly6C+ monocytes, but in the colon only by the latter demonstrating differential tolerance mechanisms along the intestinal tract. Consistently, depletion of both cell subsets reduced small intestinal POI, whereas the depletion of Ly6C+ monocytes alone was sufficient to prevent large intestinal POI. The differential role of monocytes and macrophages in small and large intestinal POI suggested a potential role of the intestinal microbiota. Indeed, antibiotic treatment reduced iNOS levels and ameliorated POI.Conclusions: Our findings reveal that CD103+CD11b+ DCs and the intestinal microbiome are a prerequisite for the activation of intestinal monocytes and macrophages and for dysregulating intestinal motility in POI

Crosstalk between sentinel and helper macrophages permits neutrophil migration into infected uroepithelium

The phagocytes of the innate immune system, macrophages and neutrophils, contribute to antibacterial defense, but their functional specialization and cooperation is unclear. Here, we report that three distinct phagocyte subsets play highly coordinated roles in bacterial urinary tract infection. Ly6C(-) macrophages acted as tissue-resident sentinels that attracted circulating neutrophils and Ly6C(+) macrophages. Such Ly6C(+) macrophages played a previously undescribed helper role: once recruited to the site of infection, they produced the cytokine TNF, which caused Ly6C(-) macrophages to secrete CXCL2. This chemokine activated matrix metalloproteinase-9 in neutrophils, allowing their entry into the uroepithelium to combat the bacteria. In summary, the sentinel macrophages elicit the powerful antibacterial functions of neutrophils only after confirmation by the helper macrophages, reminiscent of the licensing role of helper T cells in antiviral adaptive immunity. These findings identify helper macrophages and TNF as critical regulators in innate immunity against bacterial infections in epithelia.publisher: Elsevier articletitle: Crosstalk between Sentinel and Helper Macrophages Permits Neutrophil Migration into Infected Uroepithelium journaltitle: Cell articlelink: http://dx.doi.org/10.1016/j.cell.2014.01.006 content_type: article copyright: Copyright © 2014 Elsevier Inc. All rights reserved.status: publishe

Structural and functional studies on ø29 DNA polymerase

The Bac i l lus subtilis phage 029 DNA polymerase, involved in protein-primed viral DNA repli- cation, contains several amino acid consensus sequences common to other eukaryotic-type DNA polymerases. Us- ing site-directed mutagenesis, we have studied the func- tional significance o f a C-terminal conserved region, rep- resented by the Lys-X-Tyr ("K-Y") motif. Single point mutants have be en constructed and the corresponding proteins have been overproduced and characterized. Measurements o f the activity o f the mutant proteins indi- cated that the invariant Lys and Tyr residues play a criti- cal role in DNA polymerization. Interestingly, substitu- tion o f the invariant Lys either by Arg or Thr, produced enzymes with an increased or a largely reduced, respec- tively, capability to use a protein as primer, an intrinsic property o f TP-priming DNA polymerases. On the other hand, the viral protein p6, whi ch stimulates initiation o f 029 DNA replication by formation o f a nucleoprotein complex at both DNA replication origins, increased (about 5-fold) the insertion fidelity o f 029 DNA polymerase during the formation o f the TP-dAMP initia- tion complex. We propose a mode l in whi ch the special strategy to maintain the integrity o f the 029 DNA ends, by means o f a "sliding-back" mechanism, could also con- tribute to increase the fidelity o f 029 DNA replication.This investigation has been aided byreseareh grant 5R01 GM27242-13 from the National Institutes o f Health, by grant n ~ PB90-0091 from Direccibn General de Investigacibn Cientifica y T6c- nica, by grant BlOT CT 91-0268 from European Economic Commani- ty, and by an institutional grant from Fundaci6n Ram6n Areces. M.A.B., J.A.E. and J.M., were predoctoral fellows from Ministerio de Educaci6n y Ciencia.Peer reviewe