SOCS2-Induced Proteasome-Dependent TRAF6 Degradation: A Common Anti-Inflammatory Pathway for Control of Innate Immune Responses

Pattern recognition receptors and receptors for pro-inflammatory cytokines provide critical signals to drive the development of protective immunity to infection. Therefore, counter-regulatory pathways are required to ensure that overwhelming inflammation harm host tissues. Previously, we showed that lipoxins modulate immune response during infection, restraining inflammation during infectious diseases in an Aryl hydrocarbon receptor (AhR)/suppressors of cytokine signaling (SOCS)2-dependent-manner. Recently, Indoleamine-pyrrole 2,3- dioxygenase (IDO)-derived tryptophan metabolites, including L-kynurenine, were also shown to be involved in several counter-regulatory mechanisms. Herein, we addressed whether the intracellular molecular events induced by lipoxins mediating control of innate immune signaling are part of a common regulatory pathway also shared by L-kynurenine exposure. We demonstrate that Tumor necrosis factor receptor-associated factor (TRAF)6 – member of a family of adapter molecules that couple the TNF receptor and interleukin-1 receptor/Toll-like receptor families to intracellular signaling events essential for the development of immune responses – is targeted by both lipoxins and L-kynurenine via an AhR/SOCS2-dependent pathway. Furthermore, we show that LXA4- and L-kynurenine-induced AhR activation, its subsequent nuclear translocation, leading SOCS2 expression and TRAF6 Lys47-linked poly-ubiquitination and proteosome-mediated degradation of the adapter proteins. The in vitro consequences of such molecular interactions included inhibition of TLR- and cytokine receptor-driven signal transduction and cytokine production. Subsequently, in vivo proteosome inhibition led to unresponsiveness to lipoxins, as well as to uncontrolled pro-inflammatory reactions and elevated mortality during toxoplasmosis. In summary, our results establish proteasome degradation of TRAF6 as a key molecular target for the anti-inflammatory pathway triggered by lipoxins and L-kynurenine, critical counter-regulatory mediators in the innate and adaptive immune systems

TRAF6 poly-ubiquitinylation and proteasomal degradation is triggered by DC exposure to LXA₄ and L-kynurenine.

<p>WT spleen-derived DCs were cultured in the presence of medium alone (M) or with LXA₄ (Lx) (1000 ng/mL), L-kynurenine (Ky) (60 µM) or IL-10 (10) (100 ng/mL) for 6 hrs. Cells were then homogenized and immuno-precipitated with an anti-TRAF6-agarose-bead mAb. The precipitates were analyzed by western blot for the presence of TRAF6, poly-ubiquitin (Lys47) and poly-ubiquitin (Lys63). (B) Total TRAF6/beta-actin western blot analysis in WT DC cell homogenates after incubation with medium alone (M), LXA₄ (Lx) (1000 ng/mL) or L-kynurenine (Ky) (60 µM) for the indicated time points. (C) Time-course, western blot detection of total TRAF6 in spleen-derived cells incubated with LXA₄ (3 nM) with or without pre-exposure to the proteasomal inhibitor PR11 (3 nM for 90 mins). (D) WT spleen-derived cells were incubated with increasing concentrations of IL-10 (black squares) or LXA₄ (red triangles) for 6 hrs, followed by fixation/permeabilization and staining for CD11c and TRAF6 and analyzed by flow cytometry. Data shown represent plotted MFI’s for TRAF6 staining in CD11c+ cells. (E) WT (squares), SOCS2-KO (triangles) or AhR-deficient (circles) spleen-derived cells were purified and incubated with medium alone or with increasing concentrations of L-kynurenine (6–60 µM) for 6 hrs, cells were then fixed, permeabilized and stained for CD11c and TRAF6. The mean fluorescence intensity of TRAF6 staining within CD11c+ cells was then analyzed by flow cytometry. (F) WT spleen-derived cells were pre-incubated with PR11 (0.03 to 3 nM), followed by incubation with medium alone (filled squares) or with LXA₄ (upright triangles –3 nM), IL-10 (inverted triangles –100 ng/mL), L-kynurenine (diamonds –60 µM), 3-OH-kynurenine (circles –3 µM) and growth hormone (empty squares –100 ng/mL) for 6 hrs. Cells were then fixed, permeabilized and stained for CD11c, CD8α and TRAF6. Cells were then analyzed by flow cytometry for their TRAF6 mean fluorescence intensity within the CD11c+CD8α+ subset. Data shown is representative of three independent experiments performed.</p

<i>socs2</i> mRNA expression in spleen-derived dendritic cells exposed to LXA₄, L-kynurenine and FICZ is AhR-dependent.

<p>WT (filled bars) or AhR-deficient (empty bars) spleen-derived dendritic cells were isolated and incubated for 4 hrs in the presence of medium alone or with LXA₄ (3 nM), L-kynurenine (60 µM) or FICZ (4 µM). Total RNA was then obtained, followed by real-time RT-PCR for <i>socs2</i>. Data shown represents expression values normalized against β-actin expression. Asterisks indicate statistically significant differences (<i>p</i><0.05) when comparing WT <i>vs.</i> AhR group samples. Data shown is representative of four independent experiments performed.</p

LXA₄ and L-kynurenine suppress pro-inflammatory gene induction in a SOCS2-dependent manner.

<p>WT (filled symbols) or SOCS2-KO (empty symbols) spleens were harvested and CD11c+ cells purified. Cells were incubated in the presence of medium alone (black squares) or with LXA₄ (3 nM – blue triangles) and L-kynurenine (60 µM – red circles) for 6 hrs, followed by stimulation with CD154, IL-1β, TNF, LPS, STAg or CpG-oligos (1 µg/mL) for 4 hrs. Cells were lized, total RNA obtained and real-time RT-PCR reaction performed for the following genes: <i>il12a</i> (A), <i>il12b</i> (B), <i>il23p19</i> (C), <i>tnf</i> (D), <i>il6</i> (E) and <i>ifna3</i> (F). Data shown represents normalized values against β-actin expression levels and are representative of three independent assays performed. Asterisks indicate statistically significant differences between the medium versus LXA₄ or L-kynurenine treated samples (<i>p</i><0.05).</p

AhR and ARNT intracellular co-localization is induced after exposure to LXA₄ and L-kynurenine.

<p>Collagenase-digested, low-density, spleen-derived cells were incubated in the presence of medium alone (yellow), or with FICZ (4 µM), LXA₄ (3 nM) or L-kynurenine (60 µM) for 150 minutes followed by staining for AhR and ARNT and analyzed using ImageStream. Data shown indicates the AhR+ARNT+ cells gated for analysis of AhR+ARNT+ staining overlay histograms. Cells analyzed were incubated with medium alone (A), LXA₄ (B), L-kynurenine (C) and FICZ (D). Panel E shows a summary of the data shown in panels A–D. Data is representative of three independent experiments performed.</p

SOCS2 and AhR are required for modulation of host immune response during infection with <i>T. gondii</i>.

<p>WT (squares), SOCS2-KO (triangles) or AhR-deficient (circles) mice (<i>n</i> = 8 mice/group) were infected i.p. with 20 cysts of <i>T. gondii</i> (ME49 strain). Animals were monitored for (A) mortality, (B) cyst formation 30 days after infection, (C) serum levels of IL-12p70 at 5 days after infection and (D) serum levels of IFN-γ at day 7 after infection. (E–F) WT (circles) or SOCS2-KO (squares) mice were infected as described above, from 12 through 30 days after infection animals received daily i.p. injection of PBS (empty symbols) or 1 µg of PR11 (filled symbols). 30 days after infection, animals (<i>n = </i>3 mice/group) were sacrificed and antigen-specific CD4+T cell IFN-γ production analyzed by intracellular cytokine staining and flow cytometry. Cells were acquired and gated for CD3+ cells, followed by analysis of the frequencies of CD4 versus IFN-γ positive cells. The frequencies of IFN-γ+ among the total CD3+CD4+ cells are shown (E). Mortality was monitored throughout the acute and early chronic infection up to 60 days after inoculation (F). Data shown is representative of at least three independent experiments performed. <i>p</i> values are indicated for statistically significant differences between WT control groups versus SOCS2-KO or AhR-deficient strains.</p

LXA₄ and L-kynurenine exposure trigger DRE-mediated gene expression.

<p>LacZ transgenic mouse spleen-derived CD11c+ cells were purified and exposed to medium alone, or with LXA₄ (3 nM), L-kynurenine (60 µM) or FICZ (4 µM) for 120 min, followed by staining with CD11c, CD8α and incubated with the beta-galactosidase substrate for 1 min at 37°C. Cells were then analyzed by flow cytometry. Data shown is representative of three independent experiments performed. Legend indicates the sample identification followed by its mean fluorescence intensity at the FL1-channel. Histograms were normalized and presented as % of maximum fluorescence, according to FlowJo analysis software.</p

LXA₄ and L-kynurenine mediate AhR nuclear translocation in spleen-derived CD11c+ cells.

<p>Collagenase-digested, low-density, spleen-derived cells were incubated in the presence of medium alone (yellow), or with FICZ (4 µM), LXA₄ (3 nM) or L-kynurenine (60 µM) for 150 minutes followed by staining for CD11c, AhR and DNA (Draq5–nuclear counterstaining) and analyzed using ImageStream. Data shown indicates the CD11c+AhR+ cells gated for analysis of Draq5+AhR+ staining overlay histograms. Cells analyzed were incubated with medium alone (A), LXA4 (B), L-kynurenine (C) and FICZ (D). Panel E shows a summary of the data shown in panels A–D. Data is representative of three independent experiments performed.</p

Lipoxin A₄ and 15-Epi-Lipoxin A₄ Protect against Experimental Cerebral Malaria by Inhibiting IL-12/IFN-γ in the Brain

<div><p>Cerebral malaria is caused by infection with <i>Plasmodium falciparum</i> and can lead to severe neurological manifestations and predominantly affects sub-Saharan African children. The pathogenesis of this disease involves unbalanced over-production of pro-inflammatory cytokines. It is clear that signaling though IL-12 receptor is a critical step for development of cerebral malaria, IL-12 genetic deficiency failed to show the same effect, suggesting that there is redundancy among the soluble mediators which leads to immunopathology and death. Consequently, counter-regulatory mediators might protect the host during cerebral malaria. We have previously showed that endogenously produced lipoxins, which are anti-inflammatory mediators generated by 5-lipoxygenase (5-LO)-dependent metabolism of arachidonic acid, limit host damage in a model of mouse toxoplasmosis. We postulated here that lipoxins might also play a counter-regulatory role during cerebral malaria. To test this hypothesis, we infected 5-LO-deficient hosts with <i>P. berghei</i> ANKA strain, which induces a mouse model of cerebral malaria (ECM). Our results show accelerated mortality concomitant with exuberant IL-12 and IFN-<b>γ</b> production in the absence of 5-lipoxygenase. Moreover, in vivo administration of lipoxin to 5-LO-deficient hosts prevented early mortality and reduced the accumulation of CD8⁺IFN-<b>γ</b>⁺ cells in the brain. Surprisingly, WT animals treated with lipoxin either at the time of infection or 3 days post-inoculum also showed prolonged survival and diminished brain inflammation, indicating that although protective, endogenous lipoxin production is not sufficient to optimally protect the host from brain damage in cerebral malaria. These observations establish 5-LO/LXA₄ as a host protective pathway and suggest a new therapeutic approach against human cerebral malaria (HCM). (255 words).</p> </div

Exogenous delivery of 15-epi-LXA₄ prolongs survival and reduces pro-inflammatory cytokine production of both WT and 5-LO-deficient mice after <i>P. berghei</i> ANKA infection.

<p>C57Bl/6 WT and <i>Alox5</i>^−/− (<i>n</i> = 8 mice/group) mice were infected i.p. with <i>P. berghei</i> ANKA strain and treated with PBS alone or with 15-epi-LXA₄ (1 µg/mouse) from day 1 to 7 after infection (shaded area in A and F) or from day 3 to 7 after infection (striped area in F). Survival was monitored (A and F) up to 20 days after inoculum. Five days after infection, mice were sacrificed and brains harvested. Tissues were homogenized, total RNA extracted and reverse transcribed for real-time RT-PCR determination of <i>il12a</i> (B and G), <i>il12b</i> (C and H), <i>ifng</i> (D and I) and <i>socs2</i> (E and J) expression. Data shown boxes/mean bar with min-max whiskers and are representative of three independent experiments performed. (*  =  <i>p</i><0.05, **  =  <i>p</i><0.01 and ***  =  <i>p</i><0.0001) as determined by One-way ANOVA with Tukey multiple comparison test.</p