Src Kinases Are Required for a Balanced Production of IL-12/IL-23 in Human Dendritic Cells Activated by Toll-Like Receptor Agonists

BACKGROUND: Pathogen recognition by dendritic cells (DC) is crucial for the initiation of both innate and adaptive immune responses. Activation of Toll-like Receptors (TLRs) by microbial molecular patterns leads to the maturation of DC, which present the antigen and activate T cells in secondary lymphoid tissues. Cytokine production by DC is critical for shaping the adaptive immune response by regulating T helper cell differentiation. It was previously shown by our group that Src kinases play a key role in cytokines production during TLR4 activation in human DC. PRINCIPAL FINDINGS: In this work we investigated the role of Src kinases during different TLRs triggering in human monocyte-derived DC (MoDC). We found that Src family kinases are important for a balanced production of inflammatory cytokines by human MoDC upon stimulation of TLR3 and 8 with their respective agonists. Disruption of this equilibrium through pharmacological inhibition of Src kinases alters the DC maturation pattern. In particular, while expression of IL-12 and other inflammatory cytokines depend on Src kinases, the induction of IL-23 and co-stimulatory molecules do not. Accordingly, DC treated with Src inhibitors are not compromised in their ability to induce CD4 T cell proliferation and to promote the Th17 subset survival but are less efficient in inducing Th1 differentiation. CONCLUSIONS: We suggest that the pharmacological modulation of DC maturation has the potential to shape the quality of the adaptive immune response and could be exploited for the treatment of inflammation-related diseases

Themis2/ICB1 Is a Signaling Scaffold That Selectively Regulates Macrophage Toll-Like Receptor Signaling and Cytokine Production

BACKGROUND: Thymocyte expressed molecule involved in selection 1 (Themis1, SwissProt accession number Q8BGW0) is the recently characterised founder member of a novel family of proteins. A second member of this family, Themis2 (Q91YX0), also known as ICB1 (Induced on contact with basement membrane 1), remains unreported at the protein level despite microarray and EST databases reporting Themis2 mRNA expression in B cells and macrophages. METHODOLOGY/PRINCIPAL FINDINGS: Here we characterise Themis2 protein for the first time and show that it acts as a macrophage signalling scaffold, exerting a receptor-, mediator- and signalling pathway-specific effect on TLR responses in RAW 264.7 macrophages. Themis2 over-expression enhanced the LPS-induced production of TNF but not IL-6 or Cox-2, nor TNF production induced by ligands for TLR2 (PAM3) or TLR3 (poly IratioC). Moreover, LPS-induced activation of the MAP kinases ERK and p38 was enhanced in cells over-expressing Themis2 whereas the activation of JNK, IRF3 or NF-kappaB p65, was unaffected. Depletion of Themis2 protein by RNA inteference inhibited LPS-induced TNF production in primary human macrophages demonstrating a requirement for Themis2 in this event. Themis2 was inducibly tyrosine phosphorylated upon LPS challenge and interacted with Lyn kinase (P25911), the Rho guanine nucleotide exchange factor, Vav (P27870), and the adaptor protein Grb2 (Q60631). Mutation of either tyrosine 660 or a proline-rich sequence (PPPRPPK) simultaneously interrupted this complex and reduced by approximately 50% the capacity of Themis2 to promote LPS-induced TNF production. Finally, Themis2 protein expression was induced during macrophage development from murine bone marrow precursors and was regulated by inflammatory stimuli both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: We hypothesise that Themis2 may constitute a novel, physiological control point in macrophage inflammatory responses

Chemical inhibition of Src family kinases affects major LPS-activated pathways in primary human macrophages.

Understanding the signalling mechanisms controlling inflammatory cytokine production is pivotal to the research of both acute and chronic immune disorders. Tyrosine phosphorylation is one of the earliest events to occur in response to an immune challenge yet the role of specific tyrosine kinases in inflammatory cytokine production has been difficult to ascribe due to conflicting literature. Here we show that the pyrazolo pyrimidine compound PP2, a selective inhibitor of Src family kinases (SFK), can inhibit LPS-induced TNF production as well as a number of other inflammatory cytokines. In addition, we show similar effects of PP2 on cytokine production when induced by other TLRs, (1, 2 and 5-8), indicating that SFK are important common regulators of TLR signalling. PP2 suppressed the activity of both TNF and IL-10 driven reporter genes, suggesting that this activity is mediated at the level of transcription. Interestingly, however, PP2 had no significant effect on the activation of NF-kappaB, or on p42/44 ERK, p46/54 JNK or p38 MAPK phosphorylation. In contrast, PP2 did inhibit AP-1 nuclear accumulation in response to LPS. Taken together, these findings show that the Src kinases are able to control inflammatory cytokine production at the transcriptional level independently of NF-kappaB, and highlight the role of the AP-1 family of transcription factors as downstream mediators of Src kinase action

Breath octane and acetaldehyde as markers for acute respiratory distress syndrome in invasively ventilated patients suspected to have ventilator-associated pneumonia

Rationale The concentration of octane and acetaldehyde in exhaled breath has good diagnostic accuracy for acute respiratory distress syndrome (ARDS). We aimed to determine whether breath octane and acetaldehyde are able to distinguish the presence and absence of ARDS in critically ill patients suspected to have ventilator-associated pneumonia (VAP). Methods This is a secondary analysis of a prospective observational study into exhaled breath analysis using gas chromatography–time-of-flight mass spectrometry. Difference in the relative abundance of octane and acetaldehyde in exhaled breath was compared between patients with and without ARDS using the Mann–Whitney U-test and the association was quantified using logistic regression. The discriminative accuracy of octane and acetaldehyde, alone or in combination, was calculated using the area under the receiver operating characteristic curve (AUROCC). Results We included 98 patients, of whom 32 had ARDS and 66 did not. The area under the acetaldehyde peak was higher in patients with ARDS (p=0.03), and associated with the presence of ARDS (OR 1.06 per 100 000 count change, 95% CI 1.02–1.13 per 100 000 count change; p=0.01). A combined model with octane and acetaldehyde showed a high specificity and low sensitivity (90% and 40.6%, respectively), with a low accuracy (AUROCC 0.65, 95% CI 0.53–0.78). Conclusion Patients suspected to have VAP with ARDS had a higher acetaldehyde concentration in exhaled breath than patients suspected to have VAP without ARDS. However, in this patient population, discrimination of these breath biomarkers for ARDS was poor, indicating the difficulty of translating diagnostic tests between clinical settings