The intestinal immune system discriminates between tolerance toward the commensal microflora and robust responses to pathogens. Maintenance of this critical balance is attributed to mucosal dendritic cells (DCs) residing in organized lymphoid tissue and dispersed in the subepithelial lamina propria. In situ parameters of lamina propria DCs (lpDCs) remain poorly understood. Here, we combined conditional cell ablation and precursor-mediated in vivo reconstitution to establish that lpDC subsets have distinct origins and functions. CD103(+) CX(3)CR1(-) lpDCs arose from macrophage-DC precursors (MDPs) via DC-committed intermediates (pre-cDCs) through a Flt3L growth-factor-mediated pathway. CD11b(+) CD14(+) CX(3)CR1(+) lpDCs were derived from grafted Ly6C(hi) but not Ly6C(lo) monocytes under the control of GM-CSF. Mice reconstituted exclusively with CX(3)CR1(+) lpDCs when challenged in an innate colitis model developed severe intestinal inflammation that was driven by graft-derived TNF-alpha-secreting CX(3)CR1(+) lpDCs. Our results highlight the critical importance of the lpDC subset balance for robust gut homeostasis.

The CX 3 C chemokine family is composed of only one member, CX 3 CL1, also known as fractalkine, which in mice is the sole ligand of the G proteincoupled, 7-transmembrane receptor CX 3 CR1. Unlike classic small peptide chemokines, CX 3 CL1 is synthesized as a membrane-anchored protein that can promote integrin-independent adhesion. Subsequent cleavage by metalloproteases, either constitutive or induced, can generate shed CX 3 CL1 entities that potentially have chemoattractive activity. To study the CX 3 C interface in tissues of live animals, we generated transgenic mice (CX 3 CL1 cherry :CX 3 CR1 gfp ), which express red and green fluorescent reporter genes under the respective control of the CX 3 CL1 and CX 3 CR1 promoters. Furthermore, we performed a structure/function analysis to differentiate the in vivo functions of membranetethered versus shed CX 3 CL1 moieties by comparing their respective ability to correct established defects in macrophage function and leukocyte survival in IntroductionChemokine (CX 3 C motif) ligand 1 (CX 3 CL1), also known as fractalkine or neurotactin, 1,2 and its receptor CX 3 CR1 3 have been assigned their own CX 3 C chemokine family. This classification is based on the 3 amino acid gap between its N-terminal cysteines in CX 3 CL1, with no spacing in CC chemokines and only one intervening amino acid in CXC chemokines. 4 CX 3 CL1 is furthermore structurally unique in that it is synthesized as a type I transmembrane protein with the CX 3 C chemokine domain presented on an extended stalk. 1,2 Both CX 3 CL1 and CX 3 CR1 are widely expressed throughout the organism; but in given tissues, expression is often highly cell type-specific. Taking advantage of mice that harbor a targeted replacement of the CX 3 CR1 gene by a GFP reporter, 5 we could, for instance, show that CX 3 CR1 expression in the brain is restricted to microglia. CX 3 CR1 expression in the gut was found limited to lamina propria macrophages and CX 3 CR1 expression in the blood is largely restricted to monocytes, which are uniform CX 3 CR1 positive, albeit with discrete expression levels. 6 CX 3 CR1 is furthermore expressed by macrophage/ dendritic cell precursors, 7 various dendritic cell (DC) progenitors, a nonclassic CD8␣ ϩ DC subset, 8 and plasmacytoid DCs. Aside from the prominent expression in the mononuclear myeloid compartment, CX 3 CR1 receptor expression has been reported for an NK cell subset 3,9 and certain T-cell populations. 3,10,11 The in vivo expression pattern of the ligand CX 3 CL1 remains less well defined and controversial 12 but has been reported for neurons, 13 intestinal epithelium, 14 and inflamed endothelium. 2 Notably, in humans eotaxin-3/CC chemokine ligand 26 was recently reported to be a functional ligand for CX 3 CR1 15 ; in mice, the CCL26 gene, however, is a pseudogene.The analysis of CX 3 C receptor and ligand knockout mice 5,16 has revealed a number of phenotypes resulting from the lack of CX 3 CR1/ CX 3 CL1 interactions. [17][18][19][20] However, in-depth knowledge of the physiologic rol...

The lamina propria that underlies and stabilizes the gut lining epithelium is densely populated with strategically located mononuclear phagocytes. Collectively, these lamina propria macrophages and dendritic cells (DC) are believed to be crucial for tissue homeostasis as well as the innate and adaptive host defense. Lamina propria DC were recently shown to gain direct access to the intestinal lumen by virtue of epithelium-penetrating dendrites. However, the role of these structures in pathogen uptake remains under debate. In this study, we report that entry of a noninvasive model pathogen (Aspergillus fumigatus conidia) into the murine small intestinal lamina propria persists in the absence of either transepithelial dendrites or lamina propria DC and macrophages. Our results suggest the existence of multiple pathogen entry pathways and point at the importance of villus M cells in the uptake of gut lumen Ags. Interestingly, transepithelial dendrites seem altogether absent from the small intestine of BALB/c mice suggesting that the function of lamina propria DC extensions resides in their potential selectivity for luminal Ags, rather than in general uptake or gut homeostasis.

Inhalational anthrax is a life-threatening infectious disease of considerable concern, especially because anthrax is an emerging bioterrorism agent. The exact mechanisms leading to a severe clinical form through the inhalational route are still unclear, particularly how immobile spores are captured in the alveoli and transported to the lymph nodes in the early steps of infection. We investigated the roles of alveolar macrophages and lung dendritic cells (LDC) in spore migration. We demonstrate that alveolar macrophages are the first cells to phagocytose alveolar spores, and do so within 10 min. However, interstitial LDCs capture spores present in the alveoli within 30 min without crossing the epithelial barrier suggesting a specific mechanism for rapid alveolus sampling by transepithelial extension. We show that interstitial LDCs constitute the cell population that transports spores into the thoracic lymph nodes from within 30 min to 72 h after intranasal infection. Our results demonstrate that LDCs are central to spore transport immediately after infection. The rapid kinetics of pathogen transport may contribute to the clinical features of inhalational anthrax.

Homing to secondary lymphoid organs and bone marrow (BM) is a central aspect of leukemic pathophysiology. We investigated the roles of the two major lymphocyte integrins LFA-1 and VLA-4 on B-cell chronic lymphocytic leukemia (CLL) cells in these processes. We found that the majority of CLL cells expressed significantly reduced LFA-1 due to low B2 integrin transcripts. VLA-4 expression was heterogenous but underwent rapid activation by the BM chemokine CXCL12. CLL cells failed to transmigrate across VCAM-1-expressing, ICAM-1-expressing, and CXCL12-expressing endothelium, whereas when LFA-1 expression was regained in subsets of CLL cells, these lymphocytes rapidly transmigrated the endothelium. Furthermore, when injected into tail veins of immunodeficient mice, normal B cells rapidly homed to lymph nodes (LN) in a LFA-1-dependent manner, whereas CLL cells did not. Nevertheless, only residual CLL subsets could reenter BM, whereas both normal and CLL cells homed to the mice spleen in an LFA-1-independent and VLA-4-independent manner. Our results suggest that CLL cells have a reduced capacity to adhere and transmigrate through multiple vascular endothelial beds and poorly home to lymphoid organs other than spleen. Integrin blocking could thus be an efficient strategy to prevent circulating CLL cells from reaching prosurvival niches in LNs and BM but not in spleen.

SummaryMastitis, an inflammatory response of the mammary tissue to invading pathogenic bacteria, is a common disease in breast-feeding women and dairy animals. Escherichia coli is a leading cause of mastitis in dairy animals. During the course of the disease the host mounts a strong inflammatory response, but specific bacterial virulence factors have not yet been identified. Here we report the use of a murine mastitis model to investigate the innate inflammatory reaction of the mammary gland. We show that lipopolysaccharide (LPS) infusion induces mastitis in wild-type mice (C3H/HeN), but not in mice expressing mutated Toll-like receptor 4 (TLR4) (C3H/HeJ). The wild-type phenotype was restored by adoptive transfer of TLR4-expressing macrophages into the alveolar milk space of C3H/HeJ mice. In contrast to the LPS treatment, infection with E. coli P4 (ECP4) resulted in inflammation even in the absence of LPS/TLR4 signalling, indicating that additional factors play a role in the pathogenesis of the intact bacteria. Furthermore, in the absence of functional TLR4 the infecting ECP4 invade the epithelial cells with high efficiency, forming intracellular microcolonies. However, adoptive transfer with TLR4-expressing macrophages drastically reduced the epithelial invasion. Taken together, these results indicate that ECP4 has an invasive potential, which is restricted by alveolar macrophages in response to the LPS/TLR4 signalling.

Natural killer (NK) cells serve as a crucial first line of defense against tumors, viral and bacterial infections. We studied the involvement of a principal activating natural killer cell receptor, natural cytotoxicity receptor 1 (NCR1), in the innate immune response to S. pneumoniae infection. Our results demonstrate that the presence of the NCR1 receptor is imperative for the early clearance of S. pneumoniae. We tied the ends in vivo by showing that deficiency in NCR1 resulted in reduced lung NK cell activation and lung IFNγ production at the early stages of S. pneumoniae infection. NCR1 did not mediate direct recognition of S. pneumoniae. Therefore, we studied the involvement of lung macrophages and dendritic cells (DC) as the mediators of NK-expressed NCR1 involvement in response to S. pneumoniae. In vitro, wild type BM-derived macrophages and DC expressed ligands to NCR1 and co-incubation of S. pneumoniae-infected macrophages/DC with NCR1-deficient NK cells resulted in significantly lesser IFNγ levels compared to NCR1-expressing NK cells. In vivo, ablation of lung macrophages and DC was detrimental to the early clearance of S. pneumoniae. NCR1-expressing mice had more potent alveolar macrophages as compared to NCR1-deficient mice. This result correlated with the higher fraction of NCR1-ligandhigh lung macrophages, in NCR1-expressing mice, that had better phagocytic activity compared to NCR1-liganddull macrophages. Overall, our results point to the essential contribution of NK-expressed NCR1 in early response to S. pneumoniae infection and to NCR1-mediated interaction of NK and S. pneumoniae infected-macrophages and -DC.

Aspergillus fumigatus is an opportunistic fungal pathogen responsible for invasive aspergillosis in immunocompromised individuals. The inefficiency of antifungal agents and high mortality rate resulting from invasive aspergillosis remain major clinical concerns. Recently, we reported on a new family of ultrashort cationic lipopeptides active in vitro against fungi. Mode of action studies supported a membranolytic or a detergent-like effect. Here, we screened several lipopeptides in vitro for their anti-A. fumigatus activity. To investigate the therapeutic properties of the selected peptides in vivo, we challenged immunosuppressed C57BL/6 wild-type mice intranasally with DsRed-labeled A. fumigatus conidia and subsequently treated the animals locally with the lipopeptides. Confocal microscopic analysis revealed the degradation of DsRed-labeled hyphal forms and residual conidia in the lungs of the mice. The most efficient peptide was tested further using a survival assay and was found to significantly prolong the life of the treated animals, whereas no mice survived with the current standard antifungal treatment with amphotericin B. Moreover, as opposed to the drug-treated lungs, the peptide-treated lungs did not display any toxicity of the peptide. Our results highlight the potential of this family of lipopeptides for the treatment of pulmonary invasive aspergillosis.Aspergillus fumigatus is a respiratory pathogen usually acquired through the inhalation of conidia reaching the small airways and the alveolar spaces. Both mucociliary clearance and phagocytic defense prevent disease development in immunocompetent individuals (24). Macrophages (M⌽) ingest and kill resting conidia mainly through nonoxidative mechanisms, while neutrophils use oxygen-dependent mechanisms to attack hyphae germinating from conidia that escape M⌽ surveillance (1,8,14). Pulmonary dendritic cells (DC) were reported to play a role in conidium/hypha internalization, transportation to the draining lymph nodes, and induction of local and peripheral Th cell reactivity to the fungus (3). The absence of these protective mechanisms in immunocompromised individuals allows conidia to germinate and invade the lung tissue (7). Invasive aspergillosis (IA) has become an increasing cause of morbidity and mortality in patients with AIDS or those undergoing allogeneic bone marrow transplantation, as well as intensive chemotherapy (1,18,19,29,31). Although newer drugs with activity against IA have reached the commercial market (4, 10), amphotericin B deoxycholate (D-AMB) is still used extensively for severe cases of IA (26). However, the reported toxicity of D-AMB remains a major concern (33). To reduce toxicity and increase dose delivery, AMB lipid formulations have been developed recently, such as liposomal AMB (L-AMB [AmBisome]) and AMB lipid complex (Abelcet) (2,15,30,32). Corresponding preclinical studies revealed an increased antifungal activity. However, this activity correlated with increased drug levels in the tissues of mouse models of invasive funga...