Production of fever mediator PGE2 in human monocytes activated with MDP adjuvant is controlled by signaling from MAPK and p300 HAT: Key role of T cell derived factor.

Fever and inflammatory responses were observed in some subjects in early clinical trials of vaccines adjuvanted with muramyl dipeptide (MDP), a NOD2 agonist. Biosynthesis of Prostaglandin E2 (PGE2) that transmits febrile signals to the brain is controlled by an inducible enzyme, Cyclooxygenase 2 (COX-2). MDP alone was not sufficient to induce expression of COX-2 and PGE2 production in vitro. Conditioned medium prepared from Peripheral Blood Mononuclear Cells (PBMCs)-derived CD3-bead purified human T cells (TCM) dramatically increased COX2 gene transcription, COX-2 protein expression, and PGE2 production in MDP-treated monocytes. We explored epigenetic changes at the COX2 promoter using Chromatin Immunoprecipitation assay (ChIP). Increase in COX2 transcription correlated with increased recruitment of RNA polymerase II (Pol II) and p300 histone acetyl transferase (HAT) to the COX2 promoter in monocytes activated with MDP and TCM. The role of p300 HAT was confirmed by using C646, an inhibitor of p300, that reduced binding of acetylated H3 and H4 histones at the COX2 promoter, COX2 transcription, and PGE2 production in monocytes. Binding of p300, Nuclear Factor Kappa B (NF-κB), and Pol II to the COX2 promoter was also sensitive to inhibitors of Mitogen-Activated Protein Kinase (MAPK) pathway and to antibodies against Macrophage-1 (Mac-1) integrin in MDP/TCM-treated monocytes. Importantly, recombinant Glycoprotein Ib alfa (GPIbα), the recently identified factor in TCM, increased binding of NF-κB, p300, and of Pol II to the COX2 promoter and COX2 transcription in MDP-treated monocytes. Our findings suggest that a second signal through Mac-1 and MAPK is triggered by a T cell derived soluble GPIbα protein leading to the assembly of the transcription machinery at the COX2 promoter and production of PGE2 in human monocytes in response to MDP/NOD2 activation.

T cell-derived soluble glycoprotein GPIbα mediates PGE2 production in human monocytes activated with the vaccine adjuvant MDP.

Vaccine adjuvants containing analogs of microbial products activate pattern recognition receptors (PRRs) on antigen-presenting cells, including monocytes and macrophages, which can cause prostaglandin E2 (PGE2) release and consequently undesired inflammatory responses and fever in vaccine recipients. Here, we studied the mechanism of PGE2 production by human monocytes activated with muramyl dipeptide (MDP) adjuvant, which activates cytosolic nucleotide-binding oligomerization domain 2 (NOD2). In rabbits, administration of MDP elicited an early increase in PGE2 followed by fever. In human monocytes, MDP alone did not induce PGE2 production. However, high amounts of PGE2 and the proinflammatory cytokines IL-1ß and IL-6 were secreted by monocytes activated with MDP in the presence of conditioned medium obtained from CD3 bead-isolated T cells (Tc CM) but not from those isolated without CD3 beads. Mass spectrometry and immunoblotting revealed that the costimulatory factor in Tc CM was glycoprotein Ib α (GPIbα). Antibody-mediated blockade of GPIbα or of its receptor, Mac-1 integrin, inhibited the secretion of PGE2, IL-1ß, and IL-6 in MDP + Tc CM-activated monocytes, whereas recombinant GPIbα protein increased PGE2 production by MDP-treated monocytes. In vivo, COX2 mRNA abundance was reduced in the liver and spleen of Mac-1 KO mice after administration of MDP compared with that of treated wild-type mice. Our findings suggest that the production of PGE2 and proinflammatory cytokines by MDP-activated monocytes is mediated by cooperation between two signaling pathways: one delivered by MDP through NOD2 and a second through activation of Mac-1 by T cell-derived GPIbα.

Differences in PGE2 production between primary human monocytes and differentiated macrophages: role of IL-1ß and TRIF/IRF3.

Prostaglandin E2 (PGE2) is induced in vivo by bacterial products including TLR agonists. To determine whether PGE2 is induced directly or via IL-1ß, human monocytes and macrophages were cultured with LPS or with Pam3CSK4 in presence of caspase-1 inhibitor, ZVAD, or IL-1R antagonist, Kineret. TLR agonists induced PGE2 in macrophages exclusively via IL-1ß-independent mechanisms. In contrast, ZVAD and Kineret reduced PGE2 production in LPS-treated (but not in Pam3CSK4-treated) monocytes, by 30-60%. Recombinant human IL-1ß augmented COX-2 and mPGES-1 mRNA and PGE2 production in LPS-pretreated monocytes but not in un-primed or Pam3CSK4-primed monocytes. This difference was explained by the finding that LPS but not Pam3CSK4 induced phosphorylation of IRF3 in monocytes suggesting activation of the TRIF signaling pathway. Knocking down TRIF, TRAM, or IRF3 genes by siRNA inhibited IL-1ß-induced COX-2 and mPGES-1 mRNA. Blocking of TLR4 endocytosis during LPS priming prevented the increase in PGE2 production by exogenous IL-1ß. Our data showed that TLR2 agonists induce PGE2 in monocytes independently from IL-1ß. In the case of TLR4, IL-1ß augments PGE2 production in LPS-primed monocytes (but not in macrophages) through a mechanism that requires TLR4 internalization and activation of the TRIF/IRF3 pathway. These findings suggest a key role for blood monocytes in the rapid onset of fever in animals and humans exposed to bacterial products and some novel adjuvants.

Use of human MonoMac6 cells for development of in vitro assay predictive of adjuvant safety in vivo.

Subunit vaccines composed of recombinant or purified antigens have a good safety record but are poorly immunogenic and require adjuvants to activate innate immunity and facilitate antigen specific immune response. Of the many adjuvant formulations that are under development, very few are licensed mainly due to concerns about adverse side effects. The goal of our study was to develop in vitro assays that could predict toxicity of adjuvants in vivo. Pro-inflammatory cytokines IL-ß, IL-6, TNF-α, and IL-8 were measured in human primary monocytes and the monocytoid cell line, MonoMac 6 (MM6), activated with a panel of TLR agonists or with adjuvants. A 0.5 EU/ml dose of Standard for endotoxin (previously shown to provide a margin between pyrogenic and non-pyrogenic substances in rabbits) was used as a comparator to establish a "safety threshold". FSL-1, Pam3CSK4, flagellin, and R848 TLR agonists but not Alum, MF59, Poly I:C, or MPL adjuvants induced cytokines in MM6 cells above the safety threshold. To confirm the predictive value of the in vitro assays, FSL-1 and flagellin were injected intramuscularly into New Zealand White (NZW) rabbits. Both TLR agonists induced fever within 6-8h post-injection followed 24-48 h later by increased C reactive protein (CRP). Importantly, an early peak in plasma prostaglandin E2 (PGE(2)) levels preceded rise in body temperature. In vitro production of PGE(2) in monocytes and MM6 cells was found following treatments with various TLR agonists but not with alum, MF59, MPL, or Poly I:C adjuvants. Together, our studies demonstrated a strong correlation between production of pro-inflammatory cytokines above a "safety threshold" and production of PGE(2)in vitro and an increase in body temperature in rabbits. The developed human cell based assays could provide an important tool for early screening of new molecular moieties and adjuvant formulations and may assist in selection of safer products.

Regeneration of the adult thymus is preceded by the expansion of K5+K8+ epithelial cell progenitors and by increased expression of Trp63, cMyc and Tcf3 transcription factors in the thymic stroma.

Studies of HIV-1-infected individuals on anti-retroviral therapies and of patients receiving lymphoablating treatments indicate that the thymus retains restorative capacity even in adults. The contributions of the thymic epithelial cells (TECs) to the regeneration of the thymus and the identity of epithelial cell progenitors were evaluated in murine models of transient thymic atrophy followed by a complete regeneration. Using microarray approach, we analyzed the pattern of gene expression in TECs sorted from mice that were depleted of thymocytes by steroid treatment or by irradiation. The initial analysis identified significant increases in the mRNA for cMyc, Trp63 and Tcf3 transcription factors known to be expressed in early epithelial cell progenitors in tissues other than the thymus. Immunohistochemistry showed that in involuted thymuses, the cMyc and Trp63 proteins were expressed in a subset of cortical thymic epithelial cells (cTECs) that were keratin 5 positive (K5(+)), typifying cTEC precursors. Importantly, confocal microscopy established that epithelial cells with the phenotype of putative TEC progenitors (i.e. K5(+)K8(+)) expressed the Trp63 protein and confirmed that K5(+)K8(+) TEC progenitors expanded significantly during atrophy and prior to the thymic regeneration. Thus, our data demonstrated for the first time that critical steps in the recovery of the adult thymus include expansion of TEC progenitors and elevated expression of Trp63, cMyc and Tcf3 transcription factors in the thymic stroma. These results suggest that TEC progenitors could be reactivated in the adult thymus and, therefore, reactivation of TEC progenitors could provide a new approach for thymic reconstitution.

Increased CXCR4-dependent HIV-1 fusion in activated T cells: role of CD4/CXCR4 association.

Activation of peripheral CD4+ T cells resulted in augmented fusion with X4 human immunodeficiency virus type 1 (HIV-1) envelope-expressing cells without parallel increases in the surface expression of CD4 or CXC chemokine receptor 4 (CXCR4). Our study used biochemical methods and biological assays to correlate the increased fusion potential of activated T cells with changes in CXCR4 isoforms and CD4-CXCR4 association. Western blot analyses of CXCR4, precipitated from resting T cells, identified several CXCR4 species with molecular weights of 47, 50, 62, and 98 kDa. After 24 h stimulation with phytohemagglutinin/interleukin-2, a marked reduction was seen in the 47-kDa, with a concomitant increase in the amounts of 50 and 62-64 kDa CXCR4. T cell activation also induced an increase in the coprecipitation of CXCR4 with CD4. The 62-kDa CXCR4 predominantly coprecipitated with CD4 and was shown to be ubiquitinated. Stripping of CD4 from the cell surface with pronase treatment prior to cell lysis only partially reduced coprecipitation of CD4 with the 62-kDa CXCR4, revealing a pool of intracellular CD4-CXCR4 complexes. Coprecipitation of CXCR4 with CD4 was reduced in activated cells treated with Brefeldin A and Monensin, suggesting that late endosomes play a role in intracellular association of CXCR4 with CD4. Confocal microscopy confirmed the colocalization of CD4 and CXCR4 within CD63+ endocytic compartments. These findings demonstrated a correlation between the enhanced susceptibility of activated T cells to HIV-1 fusion and accumulation of ubiquitinated 62-64 kDa CXCR4 species, which preferentially associated with CD4. The CD4-CXCR4 complexes may shuttle between late endosomes and the cell surface.

CXCR4 heterogeneity in primary cells: possible role of ubiquitination.

The chemokine receptor CXCR4 is a primary coreceptor for the HIV-1 virus. The predicted molecular weight (MW) of glycosylated CXCR4 is 45-47 kDa. However, immunoblots of whole cell lysates from human lymphocytes, monocytes, macrophages, and the Jurkat T-lymphocyte line revealed multiple MW isoforms of CXCR4. Three of the bands could be precipitated by anti-CXCR4 monoclonal antibodies (101 and 47 kDa) or coprecipitated with CD4 (62 kDa). Expression of these isoforms was enhanced by infection with a recombinant vaccinia virus encoding CXCR4. In immunoblots of two-dimensional gels, antiubiquitin antibodies reacted with the 62-kDa CXCR4 species from monocytes subsequent to coprecipitation with anti-CD4 antibodies. Culturing of monocytes and lymphocytes with lactacystin enhanced the amount of the 101-kDa CXCR4 isoform in immunoblots by three- to sevenfold. In lymphocytes, lactacystin also increased cell-surface expression of CXCR4, which correlated with enhanced fusion with HIV-1 envelope-expressing cells. Similar increases in the intensity of the 101-kDa isoform were seen after treatment with the lysosomal inhibitors monensin and ammonium chloride. Antiubiquitin antibodies reacted with multiple proteins above 62 kDa, which were precipitated with anti-CXCR4 antibodies. Our data indicate that ubiquitination may contribute to CXCR4 heterogeneity and suggest roles for proteasomes and lysosomes in the constitutive turnover of CXCR4 in primary human cells.