Functional Identification of Dendritic Cells in the Teleost Model, Rainbow Trout (Oncorhynchus mykiss)

Dendritic cells are specialized antigen presenting cells that bridge innate and adaptive immunity in mammals. This link between the ancient innate immune system and the more evolutionarily recent adaptive immune system is of particular interest in fish, the oldest vertebrates to have both innate and adaptive immunity. It is unknown whether dendritic cells co-evolved with the adaptive response, or if the connection between innate and adaptive immunity relied on a fundamentally different cell type early in evolution. We approached this question using the teleost model organism, rainbow trout (Oncorhynchus mykiss), with the aim of identifying dendritic cells based on their ability to stimulate naïve T cells. Adapting mammalian protocols for the generation of dendritic cells, we established a method of culturing highly motile, non-adherent cells from trout hematopoietic tissue that had irregular membrane processes and expressed surface MHCII. When side-by-side mixed leukocyte reactions were performed, these cells stimulated greater proliferation than B cells or macrophages, demonstrating their specialized ability to present antigen and therefore their functional homology to mammalian dendritic cells. Trout dendritic cells were then further analyzed to determine if they exhibited other features of mammalian dendritic cells. Trout dendritic cells were found to have many of the hallmarks of mammalian DCs including tree-like morphology, the expression of dendritic cell markers, the ability to phagocytose small particles, activation by toll-like receptor-ligands, and the ability to migrate in vivo. As in mammals, trout dendritic cells could be isolated directly from the spleen, or larger numbers could be derived from hematopoietic tissue and peripheral blood mononuclear cells in vitro

CX3CR1+ Cell–Mediated Salmonella Exclusion Protects the Intestinal Mucosa during the Initial Stage of Infection

During Salmonella Typhimurium infection, intestinal CX3CR1(+) cells can either extend transepithelial cellular processes to sample luminal bacteria or, very early after infection, migrate into the intestinal lumen to capture bacteria. However, until now, the biological relevance of the intraluminal migration of CX3CR1(+) cells remained to be determined. We addressed this by using a combination of mouse strains differing in their ability to carry out CX3CR1-mediated sampling and intraluminal migration. We observed that the number of S. Typhimurium traversing the epithelium did not differ between sampling-competent/migration-competent C57BL/6 and sampling-deficient/migration-competent BALB/c mice. In contrast, in sampling-deficient/migration-deficient CX3CR1(-/-) mice the numbers of S. Typhimurium penetrating the epithelium were significantly higher. However, in these mice the number of invading S. Typhimurium was significantly reduced after the adoptive transfer of CX3CR1(+) cells directly into the intestinal lumen, consistent with intraluminal CX3CR1(+) cells preventing S. Typhimurium from infecting the host. This interpretation was also supported by a higher bacterial fecal load in CX3CR1(+/gfp) compared with CX3CR1(gfp/gfp) mice following oral infection. Furthermore, by using real-time in vivo imaging we observed that CX3CR1(+) cells migrated into the lumen moving through paracellular channels within the epithelium. Also, we reported that the absence of CX3CR1-mediated sampling did not affect Ab responses to a noninvasive S. Typhimurium strain that specifically targeted the CX3CR1-mediated entry route. These data showed that the rapidly deployed CX3CR1(+) cell-based mechanism of immune exclusion is a defense mechanism against pathogens that complements the mucous and secretory IgA Ab-mediated system in the protection of intestinal mucosal surface

Chemokine (C-C Motif) Receptor 2 Mediates Dendritic Cell Recruitment to the Human Colon but Is Not Responsible for Differences Observed in Dendritic Cell Subsets, Phenotype, and Function Between the Proximal and Distal Colon.

BACKGROUND & AIMS: Most knowledge about gastrointestinal (GI)-tract dendritic cells (DC) relies on murine studies where CD103+ DC specialize in generating immune tolerance with the functionality of CD11b+/- subsets being unclear. Information about human GI-DC is scarce, especially regarding regional specifications. Here, we characterized human DC properties throughout the human colon. METHODS: Paired proximal (right/ascending) and distal (left/descending) human colonic biopsies from 95 healthy subjects were taken; DC were assessed by flow cytometry and microbiota composition assessed by 16S rRNA gene sequencing. RESULTS: Colonic DC identified were myeloid (mDC, CD11c+CD123-) and further divided based on CD103 and SIRPα (human analog of murine CD11b) expression. CD103-SIRPα+ DC were the major population and with CD103+SIRPα+ DC were CD1c+ILT3+CCR2+ (although CCR2 was not expressed on all CD103+SIRPα+ DC). CD103+SIRPα- DC constituted a minor subset that were CD141+ILT3-CCR2-. Proximal colon samples had higher total DC counts and fewer CD103+SIRPα+ cells. Proximal colon DC were more mature than distal DC with higher stimulatory capacity for CD4+CD45RA+ T-cells. However, DC and DC-invoked T-cell expression of mucosal homing markers (β7, CCR9) was lower for proximal DC. CCR2 was expressed on circulating CD1c+, but not CD141+ mDC, and mediated DC recruitment by colonic culture supernatants in transwell assays. Proximal colon DC produced higher levels of cytokines. Mucosal microbiota profiling showed a lower microbiota load in the proximal colon, but with no differences in microbiota composition between compartments. CONCLUSIONS: Proximal colonic DC subsets differ from those in distal colon and are more mature. Targeted immunotherapy using DC in T-cell mediated GI tract inflammation may therefore need to reflect this immune compartmentalization

Ly6Chi monocyte recruitment is responsible for Th2 associated host-protective macrophage accumulation in liver inflammation due to schistosomiasis

Accumulation of M2 macrophages in the liver, within the context of a strong Th2 response, is a hallmark of infection with the parasitic helminth, Schistosoma mansoni, but the origin of these cells is unclear. To explore this, we examined the relatedness of macrophages to monocytes in this setting. Our data show that both monocyte-derived and resident macrophages are engaged in the response to infection. Infection caused CCR2-dependent increases in numbers of Ly6Chi monocytes in blood and liver and of CX3CR1+ macrophages in diseased liver. Ly6Chi monocytes recovered from liver had the potential to differentiate into macrophages when cultured with M-CSF. Using pulse chase BrdU labeling, we found that most hepatic macrophages in infected mice arose from monocytes. Consistent with this, deletion of monocytes led to the loss of a subpopulation of hepatic CD11chi macrophages that was present in infected but not naïve mice. This was accompanied by a reduction in the size of egg-associated granulomas and significantly exacerbated disease. In addition to the involvement of monocytes and monocyte-derived macrophages in hepatic inflammation due to infection, we observed increased incorporation of BrdU and expression of Ki67 and MHC II in resident macrophages, indicating that these cells are participating in the response. Expression of both M2 and M1 marker genes was increased in liver from infected vs. naive mice. The M2 fingerprint in the liver was not accounted for by a single cell type, but rather reflected expression of M2 genes by various cells including macrophages, neutrophils, eosinophils and monocytes. Our data point to monocyte recruitment as the dominant process for increasing macrophage cell numbers in the liver during schistosomiasis

Identification and Characterization of Specialized Antigen Presenting Cells in Rainbow Trout

As the oldest vertebrates to possess intact innate and adaptive immune systems fish are the ideal models to study evolution of adaptive immunity. With the advent of adaptive immunity came a new challenge: the coordination of temporally and spatially diverse responses to pathogens. In mammals, dendritic cells (DCs), as a result of their distinctive activation program and specialization in antigen presentation, function as a link between innate and adaptive immunity. The question as to whether dendritic cells arose concurrently with adaptive immunity or at a later date is unknown. It is possible that antigen presentation in fish is primarily accomplished by another cell type or types. The goal of my research was to identify the functional equivalent of mammalian dendritic cells in fish. In order to do this, significant numbers of cells were needed to enable functional characterization of presumptive dendritic cells. Dendritic cells are a rare cell type and in order to generate adequate numbers of cells for in depth analysis, protocols have been developed in mammals to culture dendritic cells. My approach to the problem, therefore, was to adapt these mammalian protocols to rainbow trout. Development of a hematopoietic culture system in rainbow trout, based on bone marrow derived DC cultures in mammals, generated DC-like cells (DCLCs) that were then characterized using criteria for classification of mammalian DCs. DCLCs resembled mammalian DCs in their expression of CD83 and MHCII, phagocytic capacity, and response to TLR-ligands; however, the most remarkable similarity was their ability to stimulate potent primary MLRs, more vigorous than those obtained with either macrophages or B-cells as stimulators. This evidence for a specialized antigen presenting cell type in rainbow trout has implications for fish vaccine development as well as comparative studies with mammals to elucidate the origins of adaptive immunity

tDCs are phagocytic.

<p>(<i>A</i>) Fluorescence of tDCs incubated for 2 hours with 1 µM opsonized fluorescent latex beads (black line) compared to tDCs alone (grey filled histogram). Gate indicates percent phagocytic cells (81.2%). Representative data are shown (mean percent phagocytic tDCs for experiment was 68.6%, n = 5). Experiment was repeated 2 times. (<i>B</i>) Cytospin showing internalized beads (beads indicated by “x”) in a representative tDC, compared to a representative macrophage (<i>C</i>) under the same conditions (too many beads to mark).</p

tDCs express MHCII and stimulate the MLR more effectively than B cells or macrophages.

<p>(<i>A</i>) tDCs are positive for surface MHCII by flow cytometry (black line histogram: MHCII hyper-immune serum, grey filled histogram: pre-immune serum control), but not IgM (<i>B,</i> black line histogram: anti-IgM, grey filled histogram: secondary antibody alone). (<i>C</i>) When compared to resting peritoneal macrophages (black dotted line histogram) and B cells (black solid line histogram), tDCs (solid grey histogram) express higher levels of MHCII on their surface. (<i>D</i>) tDCs were cultured with CFSE stained spleen responders in a primary allogeneic MLR at tDC to responder ratios from 1∶1 to 1∶8. Numbers represent percent dividing responders. (<i>E</i>) Allogeneic primary MLRs were conducted with tDCs, macrophages, or B cells as stimulators. Percent dividing cells was determined by flow cytometry analysis of CFSE dilution. Data are representative of 3 independent experiments, each using stimulators and responders from multiple individual fish.</p

DC marker expression.

<p>RT-PCR was performed for trout homologs of mammalian DC markers of interest. (<i>A</i>) tDCs express mRNA for TLR-3, -5,-9,-20,-22, and-22L. (<i>B</i>) tDCs express mRNA for the B7 costimulatory molecules B7R, B7H1, B7H3, and B7H4. (<i>C</i>) tDCs express mRNA for molecules involved in DC function including: IL12p40, CXCR4, CCR7, MHCII, CD83, and CD209. Results for tDCs from six individual fish are shown. PCRs shown were performed in parallel. No PCR products were detected in no RT controls or no template controls (GPDH no RT controls shown, other data not shown). (<i>D</i>) Quantitative real-time RT-PCR analysis of CD83 and MHCII mRNA expression in tDCs compared to muscle (error bars indicate SD, *p<0.05).</p

Culture and enrichment of tDCs.

<p>Phase bright (<i>A</i>) and phase contrast (<i>B</i>) images of hematopoietic cultures showing non-adherent cells with dendritic morphology. Flow cytometry forward/side scatter profile of cultured cells before and after enrichment (<i>C</i>). Gates indicate tDC population; typical purity after enrichment was 70–80%. (<i>D</i>) Low magnification of Giemsa-Wright stained cytospin of enriched cells. (<i>E</i>) High magnification of Giemsa-Wright stained cytospin of enriched cells.</p

Activation of tDCs by TLR-ligands.

<p>Phase contrast images show aggregation of tDCs typical of activated leukocytes after 24 hrs incubation with TLR-ligands (<i>A</i>), while tDCs in medium alone remained randomly distributed (<i>B</i>). tDC CD83 mRNA copy numbers, measured by real-time RT-PCR, increase significantly (*p<0.05) compared to media controls at 24 hrs of exposure to TLR-ligands (<i>C</i>), while MHCII mRNA copy numbers are not significantly changed (<i>D</i>). However, after 4 day incubation with TLR-ligands, tDCs upregulate surface expression of MHCII (<i>E</i>), (grey dotted histogram: TLR-ligand treated tDCs, black line histogram: media control tDCS, and grey filled histogram: pre-immune serum control) and by cytospin take on a morphology strikingly similar to mammalian DCs (<i>F</i>).</p