Christopher D. Jenkins

Relationships between Distinct Blood Monocyte Subsets and Migrating Intestinal Lymph Dendritic Cells In Vivo under Steady-State Conditions

The origins of dendritic cells (DCs) are poorly understood. In inflammation, DCs can arise from blood monocytes (MOs), but their steady-state origin may differ, as shown for Langerhans cells. Two main subsets of MOs, defined by expression of different chemokine receptors, CCR2 and CX3CR1, have been described in mice and humans. Recent studies have identified the inflammatory function of CCR2highCX3CR1low MOs but have not defined unambiguously the origin and fate of CCR2lowCX3CR1high cells. In this study, we show that rat MOs can also be divided into CCR2highCX3CR1low(CD43low) and CCR2lowCX3CR1high(CD43high) subsets with distinct migratory properties in vivo. Using whole body perfusion to obtain MOs, including the marginating pool, we show by adoptive transfer that CD43low MOs can differentiate into CD43high MOs in blood without cell division. By adoptive transfer of blood MOs followed by collection of pseudoafferent lymph, we show for the first time that a small proportion of intestinal lymph DCs are derived from CCR2lowCX3CR1high(CD43high) blood MOs in vivo under steady-state conditions. This study confirms one of the possible origins of CCR2lowCX3CR1high blood MOs and indicate that they may contribute to migratory intestinal DCs in vivo in the absence of inflammatory stimuli.

Intestinal Dendritic Cell Subsets: Differential Effects of Systemic TLR4 Stimulation on Migratory Fate and Activation In Vivo

Dendritic cells (DC) present peripheral Ags to T cells in lymph nodes, but also influence their differentiation (tolerance/immunity, Th1/Th2). To investigate how peripheral conditions affect DC properties and might subsequently regulate T cell differentiation, we examined the effects of a potent DC-activating, TLR-4-mediated stimulus, LPS, on rat intestinal and hepatic DC in vivo. Steady-state rat intestinal and hepatic lymph DC are αE2 integrinhigh (CD103) and include two subsets, signal regulatory protein α (SIRPα)hi/low, probably representing murine CD8αα−/+ DC. Steady-state lamina propria DC are immature; surface MHC class IIlow, but steady-state lymph DC are semimature, MHC class IIhigh, but CD80/86low. Intravenous LPS induced rapid lamina propria DC emigration and increased lymph DC traffic without altering SIRPαhigh/SIRPαlow proportions. CD80/86 expression on lymph or mesenteric node DC was not up-regulated after i.v. LPS. In contrast, i.v. LPS stimulated marked CD80/86 up-regulation on splenic DC. CD80/86 expression on intestinal lymph DC, however, was increased after in vitro culture with TNF-α or GM-CSF, but not with up to 5 μg/ml LPS. Steady-state SIRPαlow DC localized to T cell areas of mesenteric nodes, spleen, and Peyer’s patch, whereas SIRPαhigh DC were excluded from these areas. Intravenous LPS stimulated rapid and abundant SIRPαhigh DC accumulation in T cell areas of mesenteric nodes and spleen. In striking contrast, i.v. LPS had no effect on DC numbers or distribution in Peyer’s patches. Our results suggest that any explanation of switching between tolerance and immunity as well as involving changes in DC activation status must also take into account differential migration of DC subsets.

Regulation of Intestinal Dendritic Cell Migration and Activation by Plasmacytoid Dendritic Cells, TNF-α and Type 1 IFNs after Feeding a TLR7/8 Ligand

Dendritic cells (DCs) migrating via lymph are the primary influence regulating naive T cell differentiation, be it active immunity or tolerance. How DCs achieve this regulation in vivo is poorly understood. Intestinal DCs are in direct contact with harmless or pathogenic luminal contents, but may also be influenced by signals from epithelial cells, macrophages, or other resident or immigrant cells. To understand the role of TLR7 and TLR8 in regulating intestinal DC function, we fed a TLR7/8 ligand (resiquimod (R-848)) to rats and mice and examined DC in pseudoafferent lymph (rat) and mesenteric lymph nodes (MLNs). Oral R-848 induced a 20- to 30-fold increase in DC output from the intestine within 10 h due to a virtually total release of lamina propria DCs. This resulted in an accumulation of DCs in the MLNs that in mice was completely TNF-α dependent. Surprisingly, intestinal lymph DCs (iL-DCs) released by R-848 did not up-regulate CD86, but did up-regulate CD25. In contrast, MLN-DCs from R-848-stimulated rats and mice expressed high levels of CD86. This DC activation in MLNs was dependent on type 1 IFNs. The major source of these rapidly released cytokines is plasmacytoid DCs (pDCs) and not classical DCs, because depletion of pDCs significantly reduces the R-848-stimulated increase in serum cytokine levels as well as the accumulation and activation of DCs in MLNs. These experiments show that TLR-mediated regulation of iL-DC functions in vivo is complex and does not depend only on direct iL-DC stimulation, but can be regulated by pDCs.

Plasmacytoid Dendritic Cells Do Not Migrate in Intestinal or Hepatic Lymph

Plasmacytoid dendritic cells (pDCs) recognize pathogen-associated molecules, particularly viral, and represent an important mechanism in innate defense. They may however, also have roles in steady-state tolerogenic responses at mucosal sites. pDCs can be isolated from blood, mucosa, and lymph nodes (LNs). Although pDCs can express peripherally derived Ags in LNs and at mucosal sites, it is not clear whether pDCs actually migrate from the periphery in lymph or whether LN pDCs acquire Ags by other mechanisms. To determine whether pDCs migrate in lymph, intestine or liver-draining LNs were removed and thoracic duct leukocytes (TDLs) were collected. TDLs expressing MHC-II and CD45R, but not TCRαβ or CD45RA, were then analyzed. These enriched TDLs neither transcribe type I IFNs nor secrete inflammatory cytokines in response to viral stimuli in vitro or after a TLR7/8 stimulus in vivo. In addition, these TDLs do not express CD5, CD90, CD200, or Siglec-H, but do express Ig, and therefore represent B cells, despite their lack of CD45RA expression. Intestinal and hepatic lymph are hence devoid of bona fide pDCs under both steady-state conditions and after TLR7/8 stimulation. This shows that any role for pDCs in Ag-specific T cell activation or tolerance must differ from the roles of classical dendritic cells, because it cannot result from peripheral Ag capture, followed by migration of pDCs via lymph to the LN.

A distinct subset of intestinal dendritic cells responds selectively to oral TLR7/8 stimulation

The intestinal innate immune system continually interacts with commensal bacteria, thus oral vaccines should induce extra/alternative activation of DC, potentially through TLR. To examine this we collected intestinal lymph DC (iL‐DC) under steady‐state conditions and after feeding resiquimod (R‐848), a synthetic TLR7/8 ligand, which we showed induces complete emptying of gut DC into lymph. iL‐DC are heterogeneous with subset‐specific functions. In this study we determined the kinetics of iL‐DC subset release, activation and cytokine secretion induced by R‐848. We show that L‐DC comprise three distinct subsets (CD172ahigh, CD172aint and CD172alow) present with similar frequencies in intestinal but not hepatic lymph. No iL‐DC express TLR7 mRNA, and only CD172a+ iL‐DC express TLR8. However, after oral R‐848 administration, output of all three subsets increases dramatically. CD172ahigh DC release precedes that of CD172alow DC, and the increased frequency of CD25high iL‐DC is restricted to the two CD172a+ subsets. After feeding R‐848 only CD172ahigh iL‐DC secrete IL‐6 and IL‐12p40. However, CD172aint and CD172ahigh DC secrete similar but markedly lower amounts when stimulated in vitro. These results highlight the importance of in vivo approaches to assess adjuvant effects on DC and give novel insights into the subset‐specific effects of an oral TLR ligand on intestinal DC.

Hyporesponsiveness of Intestinal Dendritic Cells to TLR Stimulation Is Limited to TLR4

Dendritic cells (DCs) are crucial to intestinal immune regulation because of their roles in inducing protective immunity against pathogens while maintaining tolerance to commensal bacteria. Nonetheless, relatively little is known about intestinal DC responsiveness to innate immune stimuli via TLRs. We have previously shown that DCs migrating from the rat intestine in lymph (iLDCs) are hyporesponsive to LPS stimulation, thus possibly preventing harmful immune responses being induced to commensal flora. In this study, to understand how iLDC function is regulated by innate immune stimuli, we have characterized the expression and function of TLRs in iLDCs isolated from the thoracic duct lymph of mesenteric lymphadenectomized rats and compared these with DCs grown from bone marrow in the presence of Flt3 ligand. We show that iLDCs express mRNAs for all TLRs, but express significantly less TLR4 mRNA than bone marrow-derived DCs. Functionally, iLDCs could be activated by TLR agonists representing intestinal pathogen-associated molecular patterns, with the important exception of the TLR4 agonist LPS. Furthermore, we show that DCs in the intestinal wall interact directly with noninvasive bacteria (Bacillus subtilis spores), leading to an increase in the output of activated iLDCs into lymph, and that DCs containing spores are activated selectively. These data highlight a functional difference between TLR4 and other TLRs. As iLDCs can respond to TLR stimulation in vitro, there must be other mechanisms that prevent their activation by commensal bacteria under steady-state conditions.

Rat bone marrow-derived dendritic cells, but not ex vivo dendritic cells, secrete nitric oxide and can inhibit T-cell proliferation

The relationships between different dendritic cell (DC) populations are not clearly established. In particular, it is not known how DC generated in vitro relate to those identified in vivo. Here we have characterized rat bone marrow‐derived DC (BMDC) and compared them with DC isolated from spleen (SDC) and pseudo‐afferent lymph (LDC). BMDC express typical DC markers and are mostly OX41 positive and CD4 negative. In contrast to ex vivo DC, some BMDC express Fc receptors. FcR+ and FcR− BMDC express similar levels of major histocompatibility complex class II molecules (MHC) and are B7 positive, but some FcR− BMDC express high levels of B7. In contrast to freshly isolated or cultured ex vivo SDC and LDC, both BMDC subpopulations can express inducible nitric oxide synthase (iNOS) and can secrete nitric oxide (NO) in amounts similar to those secreted by peritoneal macrophages. Despite expressing MHC class II and B7, FcR+ BMDC stimulate only a very weak MLR and inhibit stimulation by FcR− BMDC and ex vivo DC. Inhibition is only partially NO dependent. FcR+ BMDC are not macrophages, as judged by adherence and phagocytosis. Both subpopulations are able to present antigen to primed T cells in vitro and are able to prime naïve CD4 T cells in vivo. However, unlike SDC, BMDC are unable to stimulate cytotoxic T‐lymphocyte (CTL) responses to a minor histocompatibility antigen. Thus, BMDC show marked differences to ex vivo DC and their relationship to those of in vivo DC populations, to date, is unclear.

Steady-state migrating intestinal dendritic cells induce potent inflammatory responses in naive CD4+ T cells.

Steady-state dendritic cells (DCs) migrating in the lymph from the intestine induce tolerance to harmless intestinal antigens, preventing inflammatory responses. To determine if such DCs are inherently tolerogenic we collected intestinal lymph DCs (L-DCs) by cannulation of the thoracic duct of rats after mesenteric lymphadenectomy, and examined their capacity to activate naive CD4+ lymphocytes in an allogeneic mixed leucocyte reaction. L-DCs stimulated strong proliferative responses, induced secretion of inflammatory cytokines including interferon-γ, and induced FoxP3-positive lymphocytes to divide. To determine if the activated CD4+ T cells had been tolerized, they were rested and restimulated with irradiated splenocytes. The restimulated CD4+ T cells again proliferated and secreted inflammatory cytokines. These data demonstrate that the DCs, which migrate from the intestine in the steady state, are paradoxically able to induce strong inflammatory responses from naive T cells, despite their role in the maintenance of oral tolerance.