Intestinal dendritic cell licensing through Toll-like receptor 4 is required for oral tolerance in allergic contact dermatitis.

BACKGROUND: Induction of oral tolerance to haptens is an efficient way to prevent allergic contact dermatitis (ACD) in mice. Toll-like receptor (TLR)-mediated sensing of the microbiota contributes to gut homeostasis, yet whether it contributes to induction of oral tolerance has not been documented. OBJECTIVE: We examined whether oral tolerance to the contact sensitizer 2,4-dinitro-fluorobenzene (DNFB) depends on microbiota/TLRs and evaluated the role of TLR4 on the tolerogenic function of intestinal dendritic cells (DCs). METHODS: Oral tolerance was induced by DNFB gavage in germ-free and mice deficient in several TLRs. Tolerance was assessed by means of suppression of contact hypersensitivity and hapten-specific IFN-γ-producing effector T cells. The tolerogenic function of intestinal DCs was tested by adoptive transfer experiments, ex vivo hapten presentation, and forkhead box p3 regulatory T-cell conversion. RESULTS: Oral tolerance induced by DNFB gavage was impaired in germ-free mice and TLR4-deficient mice. Bone marrow chimeras revealed that TLR4 expression on hematopoietic cells was necessary for oral tolerance induction. TLR4 appeared to be essential for the ability of intestinal dendritic cells from DNFB-fed mice to inhibit ACD on adoptive transfer. Indeed, TLR4 conditioned the in vivo mobilization to mesenteric lymph nodes of intestinal migratory CD103+ DCs carrying oral DNFB, especially the CD103+CD11b+ DC subset expressing the vitamin A-converting enzyme retinaldehyde dehydrogenase and specialized in forkhead box p3-positive regulatory T-cell conversion. CONCLUSIONS: Our data demonstrate that TLR4 conditions induction of oral tolerance to DNFB through licensing tolerogenic gut DCs. Oral biotherapy with TLR4 ligands might be useful to potentiate oral tolerance to haptens and alleviate ACD in human subjects.

Mouse and Human Liver Contain Immunoglobulin A-Secreting Cells Originating From Peyer's Patches and Directed Against Intestinal Antigens.

BACKGROUND & AIMS: The liver receives blood from the gastrointestinal tract through the portal vein, and thereby is exposed continuously to dietary antigens and commensal bacteria. Alcoholic liver disease (ALD) is associated with intestinal dysbiosis, increased intestinal permeability, release of microbes into the portal circulation, and increased serum levels and liver deposits of IgA. We characterized B-cell production of IgA in livers of mice at homeostasis, after oral immunization, in a mouse model of ALD and in human liver samples. METHODS: We performed studies with Balb/c and C57BL/6-Ly5.1 mice, as well as transgenic mice (quasimonoclonal, activation-induced [cytidine] deaminase-Cre-tamoxifen-dependent estrogen receptor 2 [ERT2], Blimp-1-green fluorescent protein [GFP]). C57BL/6-Ly5.1 mice were fed chronic plus binge ethanol to create a model of ALD. Some mice also were given repeated injections of FTY720, which prevents egress of IgA-secreting cells from Peyer's patches. We obtained nontumor liver tissues from patients with colorectal carcinoma undergoing surgery for liver metastases or hepatocellular carcinoma. B cells were isolated from mouse and human liver tissues and analyzed by flow cytometry and enzyme-linked ImmunoSpot (ELISpot). In wild-type and transgenic mice, we traced newly generated IgA-secreting cells at steady state and after oral immunization with 4-hydroxy-3-nitrophenylacetyl (NP)-Ficoll or cholera toxin. IgA responses were also evaluated in our model of ALD. RESULTS: Livers of control mice contained proliferative plasmablasts that originated from Peyer's patches and produced IgAs reactive to commensal bacteria. After oral immunization with cholera toxin or a thymus-independent antigen, a substantial number of antigen-specific IgA-secreting cells was found in the liver. Mice fed ethanol had features of hepatitis and increased numbers of IgA-secreting cells in liver, compared with mice given control diets, as well as higher levels of serum IgA and IgA deposits in liver sinusoids. Injection of FTY720 during ethanol feeding reduced liver and serum levels of IgA and IgA deposits in liver and prevented liver injury. Human liver tissues contained a significant proportion of IgA-producing plasma cells that shared phenotypic and functional attributes with those from mouse liver, including reactivity to commensal bacteria. CONCLUSIONS: Based on studies of mice and human liver tissues, we found the liver to be a site of IgA production by B cells, derived from gut-associated lymphoid tissues. These IgAs react with commensal bacteria and oral antigens. Livers from mice with ethanol-induced injury contain increased numbers of IgA-secreting cells and have IgA deposits in sinusoids. IgAs in the liver could mediate clearance of gut-derived antigens that arrive through portal circulation at homeostasis and protect these organs from pathogens.

Plasmacytoid dendritic cells are dispensable for noninfectious intestinal IgA responses in vivo.

Intestinal DCs orchestrate gut immune homeostasis by dampening proinflammatory T-cell responses and inducing anti-inflammatory IgA responses. Although no specific DC subset has been strictly assigned so far to govern IgA response, some candidate subsets emerge. In particular, plasmacytoid DCs (pDCs), which notoriously promote anti-viral immunity and T-cell tolerance to innocuous antigens (Ags), contribute to IgA induction in response to intestinal viral infection and promote T-cell-independent IgA responses in vitro. Here, using two transgenic mouse models, we show that neither short-term nor long-term pDC depletion alters IgA class switch recombination in Peyer's patches and frequency of IgA plasma cells in intestinal mucosa at steady state, even in the absence of T-cell help. In addition, pDCs are dispensable for induction of intestinal IgA plasma cells in response to oral immunization with T-cell-dependent or T-cell-independent Ags, and are not required for proliferation and IgA switch of Ag-specific B cells in GALT. These results show that pDCs are dispensable for noninfectious IgA responses, and suggest that various DC subsets may play redundant roles in the control of intestinal IgA responses.

Development of a Model for Chemical Screening Based on Collateral Sensitivity to Target BTK C481S Mutant.

Targeted therapies have improved the outcome of cancer, but their efficacy is intrinsically limited by the emergence of subclones with a mutation in the gene encoding the target protein. A few examples of collateral sensitivity have demonstrated that the conformational changes induced by these mutations can create unexpected sensitivity to other kinase inhibitors, but whether this concept can be generalized is unknown. Here is described the development of a model to screen a library of kinase inhibitors for collateral sensitivity drugs active on the Bruton Tyrosine Kinase (BTK) protein with the ibrutinib resistance mutation C481S. First, we demonstrate that overexpression of the constitutively active mutant of BTK harboring the E41K mutation in Ba/F3 cells creates an oncogenic addiction to BTK. Then, we have exploited this phenotype to perform a screen of a kinase inhibitor library on cells with or without the ibrutinib resistance mutation. The BTK inhibitors showed the expected sensitivity profile, but none of the drugs tested had a specific activity against the C481S mutant of BTK, suggesting that extending the collateral sensitivity paradigm to all kinases targeted by cancer therapy might not be trivial.

Gut Inflammation in Mice Triggers Proliferation and Function of Mucosal Foxp3+ Regulatory T Cells but Impairs Their Conversion from CD4+ T Cells.

BACKGROUND AND AIMS: Regulatory Foxp3+CD4+ T cells [Tregs] have been implicated in the control of colitis in T-cell transfer models, yet their ability to regulate colitis induced by innate immunity and the impact of gut inflammation on their fate and function have been poorly documented. METHODS: Colitis was induced by dextran sodium sulphate in DEREG transgenic mice. Tregs ablation and transfer experiments showd that Tregs could limit the severity of colitis in B6 mice. RESULTS: Gut inflammation resulted in increased number of Tregs in mesenteric lymph nodes [MLN] and colon lamina propria [LP], although their frequency decreased due to massive concomitant leukocyte infiltration. This coincided at both sites with a dramatic increase in Ki67+ Tregs which retained proliferative capacity. Gut inflammation resulted in enhanced suppressive function of Tregs in colon lamina propria and neuropillin-1- [NRP1-] Treg in MLN. Real-time polymerase chain reaction analysis and flow cytometry [using IL10-egfp-reporter mice] showed that compared with NRP1+ Treg, NRP1- Treg express higher levels of IL-10 transcripts and were enriched in IL10-expressing cells both in the steady state and during colitis. Moreover, Treg conversion in vivo from from naïve CD4+ T cells or Treg precursors was impaired in colitic mice. Finally, gut inflammation caused a decrease in intestinal dendritic cells, affecting both CD103+CD11b+ and CD103+CD11b- subsets and affected their Treg conversion capacity. CONCLUSIONS: Together, our data indicate that non-specific colon inflammation triggers proliferation and suppressive function of Tregs in the lamina propria and MLN, but impairs their de novo conversion from CD4+ T cells by intestinal dendritic cells.

Mature IgM-expressing plasma cells sense antigen and develop competence for cytokine production upon antigenic challenge.

Dogma holds that plasma cells, as opposed to B cells, cannot bind antigen because they have switched from expression of membrane-bound immunoglobulins (Ig) that constitute the B-cell receptor (BCR) to production of the secreted form of immunoglobulins. Here we compare the phenotypical and functional attributes of plasma cells generated by the T-cell-dependent and T-cell-independent forms of the hapten NP. We show that the nature of the secreted Ig isotype, rather than the chemical structure of the immunizing antigen, defines two functionally distinct populations of plasma cells. Fully mature IgM-expressing plasma cells resident in the bone marrow retain expression of a functional BCR, whereas their IgG+ counterparts do not. Antigen boost modifies the gene expression profile of IgM+ plasma cells and initiates a cytokine production program, characterized by upregulation of CCL5 and IL-10. Our results demonstrate that IgM-expressing plasma cells can sense antigen and acquire competence for cytokine production upon antigenic challenge.