Leukotriene B4 enhances the generation of proinflammatory microRNAs to promote MyD88-dependent macrophage activation.

MicroRNAs are known to control TLR activation in phagocytes. We have shown that leukotriene (LT) B4 (LTB4) positively regulates macrophage MyD88 expression by decreasing suppressor of cytokine signaling-1 (SOCS-1) mRNA stability. In this study, we investigated the possibility that LTB4 control of MyD88 expression involves the generation of microRNAs. Our data show that LTB4, via its receptor B leukotriene receptor 1 (BLT1) and Gαi signaling, increased macrophage expression of inflammatory microRNAs, including miR-155, miR-146b, and miR-125b. LTB4-mediated miR-155 generation was attributable to activating protein-1 activation. Furthermore, macrophage transfection with antagomirs against miR-155 and miR-146b prevented both the LTB4-mediated decrease in SOCS-1 and increase in MyD88. Transfection with miR-155 and miR-146b mimics decreased SOCS-1 levels, increased MyD88 expression, and restored TLR4 responsiveness in both wild type and LT-deficient macrophages. To our knowledge, our data unveil a heretofore unrecognized role for the GPCR BLT1 in controlling expression of microRNAs that regulate MyD88-dependent activation of macrophages.

PPAR-γ/IL-10 axis inhibits MyD88 expression and ameliorates murine polymicrobial sepsis.

Polymicrobial sepsis induces organ failure and is accompanied by overwhelming inflammatory response and impairment of microbial killing. Peroxisome proliferator-activated receptor (PPAR)-γ is a nuclear receptor with pleiotropic effects on lipid metabolism, inflammation, and cell proliferation. The insulin-sensitizing drugs thiazolidinediones (TZDs) are specific PPAR-γ agonists. TZDs exert anti-inflammatory actions in different disease models, including polymicrobial sepsis. The TZD pioglitazone, which has been approved by the U.S. Food and Drug Administration, improves sepsis outcome; however, the molecular programs that mediate its effect have not been determined. In a murine model of sepsis, we now show that pioglitazone treatment improves microbial clearance and enhances neutrophil recruitment to the site of infection. We also observed reduced proinflammatory cytokine production and high IL-10 levels in pioglitazone-treated mice. These effects were associated with a decrease in STAT-1-dependent expression of MyD88 in vivo and in vitro. IL-10R blockage abolished PPAR-γ-mediated inhibition of MyD88 expression. These data demonstrate that the primary mechanism by which pioglitazone protects against polymicrobial sepsis is through the impairment of MyD88 responses. This appears to represent a novel regulatory program. In this regard, pioglitazone provides advantages as a therapeutic tool, because it improves different aspects of host defense during sepsis, ultimately enhancing survival.

STAT3 Signaling Heterogeneity Underlies Cytokine-Expressing Fate in Th17 Cultures.

The polarization of naive Th cells into differentiated subsets in vitro was a powerful approach to define the development and function of Th cells in vivo. Th cell cultures identified cytokines that promote polarization and defined the phenotype and stability of differentiated cells. One of the limitations of this approach is the heterogeneity of the differentiated culture, essentially with regard to what proportion of the culture is secreting the hallmark cytokine of interest. This heterogeneity has always been puzzling because all cells in the culture have been exposed to identical culture conditions. We examined this phenomenon using an Il17f lineage-tracing allele (Cost, Cre on seventeen transcript) crossed to stop-flox Rosa-YFP (yellow fluorescent protein) mice. We found that less than half of the cells in a Th17 culture become lineage-positive during a differentiation culture and that it is primarily cells that are lineage-positive that produce cytokines when cultures are restimulated after differentiation. We sorted and analyzed YFP-positive and YFP-negative cells and found similar expression of many Th17 transcription factors, although YFP-negative cells had increased expression of other lineage-defining transcription factors. We observed that YFP-negative cells had diminished expression of Stat3 and Il6ra, as well as decreased STAT3 activation. YFP-negative cells transduced with active STAT3 had significant increases in IL-17A expression, without increases in Th17 transcription factors. Taken together, these data suggest that there is a threshold of STAT3 activation that is required for efficient Th17 differentiation, and that even in a culture of homogeneous naive T cells there is heterogeneity in the receipt of early cytokine signals.

43 kDa Glycoprotein (gp43) from Paracoccidioides brasiliensis Induced IL-17A and PGE2 Production by Human Polymorphonuclear Neutrophils: Involvement of TLR2 and TLR4.

The glycoprotein gp43 is the major antigenic/diagnostic component of Paracoccidioides brasiliensis, one of the etiologic agents of paracoccidioidomycosis (PCM). Gp43 has protective roles in mice, but due to adhesive properties, this glycoprotein has also been associated with immune evasion mechanisms. The present study evaluated gp43 interaction in vitro with Toll-like receptors 2 and 4 (TLR2 and TLR4) present in polymorphonuclear neutrophils (PMNs) from healthy human individuals and the consequent modulation of the immune response through the expression and release of cytokines and eicosanoids. PMNs were incubated in the absence or presence of monoclonal antibodies anti-TLR2 and anti-TLR4 (individually or in combination) before gp43 stimulation. Then, PMNs were analyzed for the expression of both surface receptors and the detection of intracytoplasmic IL-17A and IL-4 using flow cytometry, while the production of PGE2, LTB4, IL-6, IL-10, IL-12, IFN-γ, and TNF-α was evaluated in the supernatants by enzyme-linked immunosorbent assay (ELISA). Our results showed that gp43 increased TLR2 and TLR4 expression by PMNs and induced PGE2 and IL-17A via TLR4 and TLR2, respectively. Thus, our data suggest that gp43 from P. brasiliensis might modulate host susceptibility to the fungal infection by affecting PGE2 and IL-17A production.