The leukocyte activation receptor CD69 controls T cell differentiation through its interaction with galectin-1

CD69 is involved in immune cell homeostasis, regulating the T cell-mediated immune response through the control of Th17 cell differentiation. However, natural ligands for CD69 have not yet been described. Using recombinant fusion proteins containing the extracellular domain of CD69, we have detected the presence of a ligand(s) for CD69 on human dendritic cells (DCs). Pulldown followed by mass spectrometry analyses of CD69-binding moieties on DCs identified galectin-1 as a CD69 counterreceptor. Surface plasmon resonance and anti-CD69 blocking analyses demonstrated a direct and specific interaction between CD69 and galectin-1 that was carbohydrate dependent. Functional assays with both human and mouse T cells demonstrated the role of CD69 in the negative effect of galectin-1 on Th17 differentiation. Our findings identify CD69 and galectin-1 to be a novel regulatory receptor-ligand pair that modulates Th17 effector cell differentiation and functionThis work was funded by grants SAF2011-25834 and ERC-2011AdG 294340-GENTRIS to F.S.-M., RECAVA RD06/0014 from the Fondo de Investigaciones Sanitarias to J.V. and F.S.-M., and INDISNET 01592006 from the Comunidad de Madrid to F.S.-M. and P.M. and by grants from the Ministerio de Economia y Competitividad (PI11/01562 to P.N.) and the Generalitat de Catalunya-AGAUR (2009SGR1409 to P.N.). The Ministry of Science and Innovation and the Pro-CNIC Foundation support CNI

HDAC6 controls innate immune and autophagy responses to TLR-mediated signalling by the intracellular bacteria Listeria monocytogenes.

Recent evidence on HDAC6 function underlines its role as a key protein in the innate immune response to viral infection. However, whether HDAC6 regulates innate immunity during bacterial infection remains unexplored. To assess the role of HDAC6 in the regulation of defence mechanisms against intracellular bacteria, we used the Listeria monocytogenes (Lm) infection model. Our data show that Hdac6-/- bone marrow-derived dendritic cells (BMDCs) have a higher bacterial load than Hdac6+/+ cells, correlating with weaker induction of IFN-related genes, pro-inflammatory cytokines and nitrite production after bacterial infection. Hdac6-/- BMDCs have a weakened phosphorylation of MAPK signalling in response to Lm infection, suggesting altered Toll-like receptor signalling (TLR). Compared with Hdac6+/+ counterparts, Hdac6-/- GM-CSF-derived and FLT3L-derived dendritic cells show weaker pro-inflammatory cytokine secretion in response to various TLR agonists. Moreover, HDAC6 associates with the TLR-adaptor molecule Myeloid differentiation primary response gene 88 (MyD88), and the absence of HDAC6 seems to diminish the NF-κB induction after TLR stimuli. Hdac6-/- mice display low serum levels of inflammatory cytokine IL-6 and correspondingly an increased survival to a systemic infection with Lm. The impaired bacterial clearance in the absence of HDAC6 appears to be caused by a defect in autophagy. Hence, Hdac6-/- BMDCs accumulate higher levels of the autophagy marker p62 and show defective phagosome-lysosome fusion. These data underline the important function of HDAC6 in dendritic cells not only in bacterial autophagy, but also in the proper activation of TLR signalling. These results thus demonstrate an important regulatory role for HDAC6 in the innate immune response to intracellular bacterial infection

Psoriasis in humans is associated with down-regulation of galectins in dendritic cells

We have investigated the expression and role of galectin-1 and other galectins in psoriasis and in the Th1/Th17 effector and dendritic cell responses associated with this chronic inflammatory skin condition. To determine differences between psoriasis patients and healthy donors, expression of galectins was analysed by RT-PCR in skin samples and on epidermal and peripheral blood dendritic cells by immunofluorescence and flow cytometry. In the skin of healthy donors, galectin-1, -3 and -9 were expressed in a high proportion of Langerhans cells. Also, galectins were differentially expressed in peripheral blood dendritic cell subsets; galectin-1 and galectin-9 were highly expressed in peripheral myeloid dendritic cells compared with plasmacytoid dendritic cells. We found that non-lesional as well as lesional skin samples from psoriasis patients had low levels of galectin-1 at the mRNA and protein levels, in parallel with low levels of IL-10 mRNA compared with skin from healthy patients. However, only lesional skin samples expressed high levels of Th1/Th17 cytokines. The analysis of galectin-1 expression showed that this protein was down-regulated in Langerhans cells and dermal dendritic cells as well as in peripheral blood CD11c + DCs from psoriasis patients. Expression of galectin-1 correlated with IL-17 and IL-10 expression and with the psoriasis area and index activity. Addition of galectin-1 to co-cultures of human monocyte-derived dendritic cells with autologous T lymphocytes from psoriasis patients attenuated the Th1 response. Conversely, blockade of galectin binding increased IFNγ production and inhibited IL-10 secretion in co-cultures of monocyte-derived dendritic cells with CD4 + T cells. Our results suggest a model in which galectin-1 down-regulation contributes to the exacerbation of the Th1/Th17 effector response in psoriasis patients.Spanish Ministry of Science and Innovation (PI080946,SAF-2008-02635,SAF-2011-25834); EU–Mexico FONCICYT (Grant No. C002-2009-1 ALA/127249); INSINET (Grant No. 01592006); MEICA (Genoma Espana)Peer Reviewe

Defective pro-inflammatory cytokine response to <i>Lm</i> in <i>Hdac6</i>^<i>-/-</i> BMDCs.

<p>A) PCR analysis of type-I interferons (PanIFN-α and IFN-β), interferon downstream proteins (Mx1, IFIT3 and ISG15), pro-inflammatory cytokines (TNF-α, IL-1β and IL-12p40) chemokine receptor (CXCR1) and chemokines (CXCL5 and CXCL10) of <i>Hdac6</i>^<i>+/+</i> and <i>Hdac6</i>^<i>-/-</i> BMDCs non-infected (NI) and infected with <i>Lm</i> at 6 hpi (arbitrary units). ***p≤0.001, ** p≤0.01, * p≤0.05; n = 5–6. B) ELISA analysis of the pro-inflammatory cytokines TNFα, IL1β, IL6 and IL12p70 (pg/ml) and IFN-β in supernatants of <i>Hdac6</i>^<i>+/+</i> and <i>Hdac6</i>^<i>-/-</i> BMDCs at 6, 12 and 24 hpi with <i>Lm</i>. ***p≤0.001, ** p≤0.01, * p≤0.05 ns>0.05 non-significant; n = 5–6.</p

<i>Hdac6</i>^<i>-/-</i> BMDCs show defective inflammatory cytokine response to Toll-like receptor stimuli.

<p>A) ELISA analysis of the pro-inflammatory cytokines TNFα, IL-1β, IL-6 and IL12p70 (pg/ml) in supernatants of <i>Hdac6</i>^<i>+/+</i> and <i>Hdac6</i>^<i>-/-</i> BMDCs after treatment for 6, 12 and 24 h with Pam3GSK4. ***p≤0.001, ** p≤0.01, * p≤0.05, ns>0.05 non-significant; n = 5–6. B) ELISA analysis of the pro-inflammatory cytokines TNFα, IL-1β, IL-6 and IL12p70 (pg/ml) in supernatants of <i>Hdac6</i>^<i>+/+</i> and <i>Hdac6</i>^<i>-/-</i> BMDCs after treatment for 6, 12 and 24 h with LPS. ***p≤0.001, ** p≤0.01, ns>0.05 non-significant; n = 5–6. C) ELISA analysis of the pro-inflammatory cytokines TNFα, IL-1β, IL-6 and IL12p70 (pg/ml) in supernatants of <i>Hdac6</i>^<i>+/+</i> and <i>Hdac6</i>^<i>-/-</i> BMDCs after treatment for 6, 12 and 24 h with Imiquimod. ***p≤0.001, * p≤0.05, ns>0.05 non-significant; n = 5–6. D) ELISA analysis of the pro-inflammatory cytokines TNFα, IL-1β, IL-6 and IL12p70 (pg/ml) in supernatants of <i>Hdac6</i>^<i>+/+</i> and <i>Hdac6</i>^<i>-/-</i> BMDCs after treatment for 6, 12 and 24 h with HKST. ***p≤0.001, ** p≤0.01, ns>0.05 non-significant; n = 5–6.</p

Association of HDAC6 with TLR-adaptor MyD88 and its contribution to the inflammatory response to <i>Lm</i>.

<p>A) ELISA analysis of the pro-inflammatory cytokines TNFα, IL-1β, IL-6 and IL12p70 (pg/ml) in supernatants of <i>Hdac6</i>^<i>+/+</i> and <i>Hdac6</i>^<i>-/-</i> FLT3L-DCs activated with LPS, Imiquimod, Pam3GSK4, HKLM, HKST, <i>Lm</i>, Poly(I:C) and flagellin for 24 h. ***p≤0.001, ** p≤0.01, * p≤0.05; n = 6. B) Immunoprecipitation of endogenous HDAC6 and MyD88 followed by western-blot for both proteins. Immunoprecipitations were carried out using human moDCs after 30 min of stimulation with Pam2GSK4, Pam3GSK4 and HKLM. Endogenous HDAC6 (130 KDa) and MyD88 (33 KDa) are indicated at right of western-blot. Similar results were obtained in two independent experiments. C) Immunoprecipitation of HA (MyD88) followed by western-blot for HDAC6 and MyD88. Immunoprecipitations were carried out using different HDAC6-eGFP plasmids co-transfected with MyD88-HA in HEK-Blue hTLR2 cell line after 30 min of stimulation with HKLM. Over-expressed (HDAC6-eGFP, 160 KDa) and endogenous HDAC6 (130 KDa) are indicated at right of western-blot. Similar results were obtained in four independent experiments. D) Immunoprecipitation of HA (MyD88) followed by mass spectrometry analysis. Immunoprecipitations were carried out using different HDAC6-eGFP plasmids co-transfected with MyD88-HA in HEK-Blue hTLR2 cell line after 30 min of stimulation with HKLM. The number of unique MyD88 and HDAC6 peptides is indicated. No acetylated peptides from MyD88 were detected in any sample. Similar results were obtained in four independent experiments. E) Graph of NF-κB induction in transfected <i>HDAC6-WT</i>, <i>HDAC6-DD</i> and <i>shHDAC6</i> HEK-Blue hTLR2 cell line after activation with HKLM, Pam2GSK4 and Pam3GSK4 during 8 h. NF-κB induction was calculated by the ratio of the signal of stimulated cells with its corresponding transfected cells in basal condition (without stimuli), ***p≤0.001, ** p≤0.01, * p≤0.05, ns>0.05 non-significant; n = 6. F) Survival curve to intravenous injection with a lethal dose of <i>Lm</i> in <i>Hdac6</i>^<i>+/+</i> and <i>Hdac6</i>^<i>-/-</i> is showed. This curve corresponds to two different experiment of survival to <i>Lm</i> with a n = 24–21. ***p≤0.001. G) Pro-inflammatory cytokine IL-6 was measured in sera of <i>Hdac6</i>^<i>+/+</i> and <i>Hdac6</i>^<i>-/-</i> mice intravenously injected with a lethal dose of <i>Lm</i> at 12, 48 and 72 hpi. *p≤0.05, n = 5.</p

Deficient intracellular bacteria clearance in <i>Hdac6</i>^<i>-/-</i> splenic myeloid populations.

<p>A) Quantification of bacterial load in target organs (spleen and liver) at 6 hpi in <i>Hdac6</i>^<i>+/+</i> and <i>Hdac6</i>^<i>-/-</i> mice injected with a lethal dose of <i>Lm</i>. Bacterial load is expressed by CFUs per gram of liver (left graph) and per gram of spleen (right graph). **p≤0.01, n = 6. B) The charts show geometric means of <i>Lm</i> of different splenic populations (monocytes, neutrophils, Tips DCs, total cDCs, cDCs CD8^- and cDCs CD8⁺) gated in the live CD3^-CD19^-DX5^- population of <i>Hdac6</i>^<i>+/+</i> and <i>Hdac6</i>^<i>-/-</i> mice injected with a lethal dose of <i>Lm</i> at 6 hpi. **p≤0.01; n = 6.</p

Defective iNOS response to <i>Lm</i> in <i>Hdac6</i>^<i>-/-</i> BMDCs.

<p>A) <i>Lm</i>-activated iNOS activity. Nitrite levels in supernatants of <i>Lm</i>-infected BMDCs at 6, 12 and 24 hpi. ***p≤0.001; n = 5. B) Western-blot analysis of iNOS induction over the time-course of <i>Lm</i> infection. β-actin was used as a loading control (top panel). The chart shows quantification of iNOS at 4 and 6 hpi. ** p≤0.01, * p≤0.05; n = 4 (lower panel). C) The panel shows representative histograms of iNOS expressed by <i>Hdac6</i>^<i>+/+</i> and <i>Hdac6</i>^<i>-/-</i> BMDCs after exposure to live <i>Lm</i> or HKLM for 24 h (left). The right chart shows the geometric mean of iNOS expression. Non-infected (NI) BMDCs were used as a control of iNOS induction. **p≤0.01; n = 6.</p