Hypo-responsiveness of intestinal dendritic cells to TLR stimulation is limited to TLR4
This very interesting paper is from the lab of Dr. Linda Klavinskis, Department of Immunology, King’s College, London.
As the intestine encounters a plethora of antigens, some of which are harmful while others are harmless, immune mechanisms have evolved to maintain immunological tolerance against harmless antigens to avoid chronic inflammation and to elicit protective responses against harmful antigens. One of the major unanswered questions in mucosal immunology is how this delicate balance is maintained in the gut.
The current literature suggests that dendritic cells (DCs) play important roles in immune regulation. It is assumed that DCs are activated on detection of microbe associated molecular patterns (MAMPs) by pathogen recognition receptors (PRRs) on DCs. Of the various PRRs, TLRs are mostly widely understood and they recognize a variety of MAMPs. However, very little is known about the capacity of intestinal DCs to detect and respond to MAMP or TLR-agonists.
Before talking about this paper, let me summarize the current knowledge on this topic. It has been observed that CD11c+ lamina propria (LP) cells (which include DCs also) were not found to be expressing TLR4 mRNA and steady state and were also found not to respond to the LPS stimulation in vitro. Similarly, mouse CD103+ mesenteric lymph nodes (MLN) DCs, which are thought to be derived from intestine, showed lower levels of TLR2 and TLR4 mRNA than CD103- MLN DCs (thought to be blood derived cells). All these studies suggest that down-regulation of TLRs on DCs could be responsible for immune tolerance to harmless antigens. However, a recent study showed that murine CD11chighCD11bhigh LP DCs express TLR5 and respond to flagellin stimulation. The authors of the current paper have also previously shown that a subset of rat intestinal lymph DCs respond to TLR7/8 agonists stimulation.
All these studies indicate that intestinal DCs might not be unresponsive to all TLR agonists and that they might express some TLRs.
The capacity of intestinal DCs to express PRRs and respond to MAMPs is a key question and its answer can aid in design of oral vaccines and provide indication of pathways that lead to inflammation. Thus, the authors in this paper sought to determine the pattern of TLR expression and functionality of intestinal DCs. The problem with the study of intestinal DCs is that the isolation procedure may itself induce phenotypic and functional changes in DCs. To overcome this problem, the authors in this paper collected lymph-born DCs from the thoracic duct lymph of mesenteric lymphadenectomized (MLNX) rats. All these intestinal lymph-borne DCs (iLDC) express high levels of CD103 and MHCII. They represent a heterogeneous population distinguished by the expression of CD172a and CD11b.
The authors showed that iLDCs were unresponsive to TLR4 agonist LPS but were able to up-regulate activation markers up on stimulation through other TLRs.
Steady State Repertoire of TLRs expressed by intestinal DCs
Experiment: iLDCs were collected from the lymph of MLNX rats. They were enriched using anti-CD-103 magnetic beads and sorted by FACS as large CD103+ MHC class II+ cells. There were three subsets of DCs. They were CD172a-, CD172a+ CD11b- and CD172a+CD11b+. On these cells the expression of mRNA of TLR1 to 11 and MD2 (a co-receptor for TLR4) was determined by qPCR and the expression levels were compared with those of Fl3l generated rat BMDCs.
Results:
1. Rat iLDC expressed mRNAs of all TLRs (1-11) except TLR7.
2. No significant difference in level of expression of TLRs 5, 6, 10, 11 and MD2 was observed in any subsets of DCs or in BMDCs.
3. The level of TLR8 mRNA was comparable in CD172a+ DCs and BMDCs.
4. The level of TLR3 mRNA expression was significantly higher in CD172a- in comparison to CD172a+ cells.
5. TLR9 was similarly expressed by all three subsets of DCs.
6. TLR4 was significantly low in all three cells than in BMDCs.
7. TLR2 levels were high on all three DCs and comparable to BMDCs.
Conclusions:
These data suggest that iLDCs express transcripts of all TLRs (except TLR7) in steady state. However, the level of expression varies in different populations of DCs.
Can intestinal DCs be activated by TLR agonists in vitro?
Experiments: Enriched iLDCs were cultured for 18hrs in the presence or absence of Pam3Cys (TLR-2 agonist), Poly (I:C) (agonist signaling through TLR3, although it can activate other cellular receptors), LPS (TLR4 agonist), flagellin (TLR5 agonist), R-848 (TLR7/8 agonist), and CpG (TLR9 agonist). The levels of activation markers CD25 and CD86 were assessed by flow cytometry.
Results: With the exception of TLR4 agonist LPS, a significant increase in percentage of cells expressing CD25 or CD86 was observed in case of culture with all other TLR ligands.
Conclusions: These data suggest that despite continuous exposure to intestinal commensal bacteria, intestinal DCs respond to TLR agonists.
Can highly pure intestinal DCs be activated by TLR agonists in vitro?
Experiments: CD103+ and MHC II+ iLDCs were sorted by FACS and cultured for 18hrs in the presence or absence of a range of TLR2 agnosits (Pam3Cys, Pam2Cys, and heat-inactivated spores of B. subtilis). The levels of activation markers CD25 and CD86 were assessed by flow cytometry. Similar experiments were repeated with TLR9 agonist CpG. Further more, the production of cytokines in response to stimulation with TLR liagnds was determined. The three populations of DCs were sorted by FACS and incubated with TLR ligands.
Results:
1. A significant increase in percentage of cells expressing CD25 or CD86 was observed in case of culture with all TLR2 and TLR9 ligands.
2. The authors also found that DCs cultured with Pam3cys, Pam2Cys, B. subtilis spores or CpG responded with enhanced secretion of IL-6 and IL-12p40 in comparison to DCs cultured alone.
3. DCs incubated with LPS did not show any enhanced secretion.
4. All three subsets of DCs secreted IL-6 and IL-12p4o inresponse to TLR2 and TLR9 ligands but not to TLR4 ligand.
5. Of the three subsets, CD172a-, but not the CD172a+CD11b- or the CD172a+CD11b+ iLDCs, secreted TNF-alpha when cultured with Pam3Cys or CpG, but not with LPS.
Intra-intestinal injection of B. subtilis spores induces a significant increase in proportion of activated DCs migrating out of intestines
Experiments: MLNX rats were injected intra-intestinally with heat inactivated spores of B. subtilis or with PBS as control. After 24 hr, thoracic duct lymph cells were collected.
Results:
1. There was no change in the proportion of intestinal DCs migrating to lymph in two groups of animals.
2. A small but significant increase in the number of DCs bearing CD25 or CD86 was detected in lymph 24 hr after injection with spores in comparison to controls.
3. Same response was observed in all three DC subsets.
Conclusions: The intra-intestinal injection of a model non-invasive Gram-positive organism causes a significant increase in up-regulation of activation markers in DCs migrating from intestine to lymph.
Do these migrating DCs directly interact with intra-intestinally injected B. subtilis spores?
Experiments: MLNX rats were injected intra-intestinally with heat inactivated CFSE labeled spores of B. subtilis or with PBS as control. After 24 hr, thoracic duct lymph cells were collected. DCs were identified as CD103+ and MHCII + cells by flow and evaluated for green fluorescence (due to CFSE) and CD25 expression. CSFE+ and CSFE- cells were sorted by FACS and mRNA levels of cytokines were examined.
Results:
1. A marked increase in green fluorescence was observed in rats injected with FCSE labeled spores in comparison to controls.
2. There was preferential activation of spore bearing DCs as the fluorescence was exclusively limited to CD25+ iLDCs.
3. CSFE+ DCs showed a 3-5 fold increase in mRNA expression of IL-6 in comparison to controls.
4. No significant increase in mRNA levels of other cytokines (IL-10, TNF-alpha, IL-12p40, IL-12p35 or IL-23p19) was observed.
5. Also, when FACS sorted CD103+ MHC II+ cells from the lymph of spore injected mice were analyzed by fluorescent confocal microscopy, bright green colored spores clearly visible within the DCs were observed.
Conclusions: There is direct interaction of intestinal DCs with B. subtilis spores which leads to activation of DCs and these spores are internalized by DCs.
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