Lack of PPARγ in Myeloid Cells Confers Resistance to Listeria monocytogenes Infection

The peroxisomal proliferator-activated receptor γ (PPARγ) is a nuclear receptor that controls inflammation and immunity. Innate immune defense against bacterial infection appears to be compromised by PPARγ. The relevance of PPARγ in myeloid cells, that organize anti-bacterial immunity, for the outcome of immune responses against intracellular bacteria such as Listeria monocytogenes in vivo is unknown. We found that Listeria monocytogenes infection of macrophages rapidly led to increased expression of PPARγ. This prompted us to investigate whether PPARγ in myeloid cells influences innate immunity against Listeria monocytogenes infection by using transgenic mice with myeloid-cell specific ablation of PPARγ (LysMCre×PPARγflox/flox). Loss of PPARγ in myeloid cells results in enhanced innate immune defense against Listeria monocytogenes infection both, in vitro and in vivo. This increased resistance against infection was characterized by augmented levels of bactericidal factors and inflammatory cytokines: ROS, NO, IFNγ TNF IL-6 and IL-12. Moreover, myeloid cell-specific loss of PPARγ enhanced chemokine and adhesion molecule expression leading to improved recruitment of inflammatory Ly6Chi monocytes to sites of infection. Importantly, increased resistance against Listeria infection in the absence of PPARγ was not accompanied by enhanced immunopathology. Our results elucidate a yet unknown regulatory network in myeloid cells that is governed by PPARγ and restrains both listeriocidal activity and recruitment of inflammatory monocytes during Listeria infection, which may contribute to bacterial immune escape. Pharmacological interference with PPARγ activity in myeloid cells might represent a novel strategy to overcome intracellular bacterial infection

The VEGF-Receptor Inhibitor Axitinib Impairs Dendritic Cell Phenotype and Function

<div><p>Inhibitors of VEGF receptor (VEGFR) signaling such as sorafenib and sunitinib that are currently used in the treatment of malignant diseases have been shown to affect immunological responses by inhibition of the function of antigen presenting cells and T lymphocytes. The VEGFR-inhibitor axitinib has recently been approved for second line therapy of metastatic renal cell carcinoma. While there is some evidence that axitinib might interfere with the activation of T cells, not much is known about the effects of axitinib on dendritic cell (DC) phenotype and function. We here show that the addition of axitinib during the final Toll-like receptor-4-induced maturation step of monocyte-derived human DCs results in a reduced DC activation characterized by impaired expression of activation markers and co-stimulatory molecules such as CD80, CD83 and CD86. We further found a decreased secretion of interleukin-12 which was accompanied by reduced nuclear expression of the transcription factor cRel. In addition, we found a dose-dependent reduced activation of p38 and STAT3 in axitinib-exposed DCs, whereas the expression was not affected. The dysfunction of axitinib-exposed DCs was further underlined by their impaired induction of allogeneic T cell proliferation in a mixed lymphocyte reaction assay and inhibition of DC migration. Our results demonstrate that axitinib significantly affects DC differentiation and function primarily via the inhibition of the nuclear factor kappa B signaling pathway leading to impaired T cell activation. This will be of importance for the design of future vaccination protocols and therapeutic approaches aiming at combining different treatment strategies, eg such as programmed death-1 inhibitors with axitinib.</p></div

Axitinib dose-dependently induces apoptosis in the renal cell carcinoma cell line A-498, but has no toxic effects on dendritic cells.

<p>Different concentrations (100 nM– 10 μM; black columns) of axitinib or the vehicle control DMSO (white column) were added to the culture of monocyte-derived DCs generated in the presence of IL-4 and GM-CSF for 5 days (A) as well as to the culture of renal cell carcinoma cell line A-498 (B). Nuclei fragmentation was analyzed 48 hours later. Results of one representative donor out of 4 are shown.</p

C-Rel, p38 and STAT3 are down-regulated in axitinib-treated moDCs.

<p>Whole cell lysates and nuclear extracts from axitinib or vehicle DMSO-treated moDCs, unstimulated or LPS-activated, were prepared and analyzed for their expression of RelA, cRel, p38, STAT3 in Western Blots. We found a dose-dependent down-regulation of cRel in axitinib-treated moDCs, whereas no significant modulation of RelA was observed in nuclear protein. The phosphorylation of p38 and STAT3 (measured in whole cell lysates) was dose-dependently decreased in higher doses, whereas the expression was not affected. HDAC1 was included as an additional loading control for nuclear protein expression. Results of one representative donor out of 3 are shown.</p

Axitinib impairs T cell stimulatory function and migratory behaviour of DCs.

<p>The ability of human moDCs, treated once with axitinib (black columns) on day 5 and activated with LPS on day 6, to prime allogeneic T cell responses <i>in vitro</i> was assessed using a mixed lymphocyte reaction assay. Irradiated stimulator DCs were cultured with responding allogeneic peripheral blood mononuclear cells. Tritium-labeled thymidine incorporation was measured 5 days later. White column represents DMSO as vehicle control, negative control represents CD3+ T cells without stimulating moDCs <b>(A)</b>. Axitinib-treated and LPS-stimulated human moDCs were assessed for their migratory behaviour towards CCL19/MIP-3β in transwell assays <b>(B)</b> and moDCs were analyzed for their surface CCR7 expression <b>(C).</b> White columns represent results of vehicle-exposed, LPS-stimulated DCs. Results are from one experiment representative of at least three. The significance was calculated according to one-way ANOVA Dunnett´s Multiple Comparison Test and is related to the vehicle control. **p<0.01.</p

Axitinib modulates cytokine levels in DCs and impairs T cell stimulatory function of DCs.

<p>Monocytes were cultured under DC-driving conditions and treated with axitinib once on day 5, followed by LPS-activation on day 6. Supernatants were collected on day 7 and analyzed for cytokine production using a commercially available FlowCytoMix Assay. Results of one representative donor out of 3 are shown.</p

Axitinib modulates DC phenotype and DC activation.

<p>Expression of DC marker CD1a, monocyte marker CD14 and activation markers CD40, CD80, CD83 and CD86 after exposure of moDCs to axitinib (100 nM, 1 μM, 5 μM and 10 μM, black columns) on day 5 or the vehicle control DMSO (white column), followed by subsequent final maturation with LPS on day 6, are shown. Results of one representative donor out of 4 are shown.</p

Enhanced effector functions following abelation of PPARγ from myeloid cells.

<p>(A) BMDM from LysM- PPARγ^WT and LysM- PPARγ^KO mice were infected <i>in vitro</i> with <i>L.monocytogenes</i> at a MOI 10. Expression of IFNγ, TNF, IL-1β, IL-6 and IL-12 was determined 6 hrs post infection in cell culture supernatant by ELISA. The data shown are means ± SE of three independent experiments. (B) Looking at the paracrine effects of PPARγ ablation in myeloid cells, we infected BMDM from wild type C57BL/6 mice with <i>Listeria</i> at an MOI of 10 and incubated them with sterile filtered conditioned medium from PPARγ^WT or PPARγ^KO macrophages infected previously for 18 hrs with <i>L. monocytogenes</i>; at the indicated time points intracellular <i>Listeria</i> growth was determined as CFU/ml in the lysates of infected cells. Experiments were performed in triplicates. One representative out of four experiments is shown. The <i>p</i> value for titers of wt vs. ko at 4 hrs post infection were <0.05.</p

Enhanced recruitment of inflammatory monocytes to the site of infection in LysM-PPARγ^KO mice.

<p>LysM- PPARγ^WT and LysM- PPARγ^KO mice were infected i.p. with <i>Listeria</i> (2×10⁴ CFU). (A) at indicated time points total numbers of CD11b⁺ Ly6C^high monocytes in the peritoneal cavity (PEC), liver and spleen were determined by FACS analysis (B–C) Expression of CCL2, CCL7 and CCR2 in the liver (B) and spleen (C) of infected mice as determined by qRT-PCR. (D) LSEC isolated from wild type C57BL/6 mice were incubated <i>in vitro</i> with sterile filtered supernatant peritoneal macrophages from LysM- PPARγ^WT and LysM- PPARγ^KO mice that were either infected (18 hrs) or left non-infected. The fold increase in expression of CD54 on the surface of LSEC determined by increase in mean fluorescence intensity by flow cytometry in response to contact with supernatant from infected vs non-infected peritoneal macrophages was determined after 24 hrs.</p

PPARγ expression is induced in bone-marrow derived monocytes after infection with <i>Listeria monocytogenes</i>.

<p>(A) Time course of PPARγ expression in human monocytes following <i>Listeria monocytogenes</i> infection detected by Western blot. (B) PPARγ target genes (n = 80) with the most significant differential expression in human macrophages post infection. Gene expression differences (log scale) are visualized as a heat map following hierarchical clustering of rows and columns (red = increased expression). (C,D) Time course of PPARγ expression in bone marrow derived macrophages (BMDM) post infection detected by Western blot or immunohistochemistry. (blue = DAPI staining the cell nucleus and <i>Listeria</i> DNA; green = PPARγ).</p