NLRP3 inflammasome pathway has a critical role in the host immunity against clinically relevant Acinetobacter baumannii pulmonary infection.

The opportunistic Gram-negative bacterium Acinetobacter baumannii (AB) is a leading cause of life-threatening nosocomial pneumonia. Outbreaks of multidrug resistant (MDR)-AB belonging to international clones (ICs) I and II with limited treatment options are major global health threats. However, the pathogenesis mechanisms of various AB clonal groups are understudied. Although inflammation-associated interleukin-1ß (IL-1ß) levels and IL-1 receptor antagonist polymorphisms were previously implicated in MDR-AB-related pneumonia in patients, whether inflammasomes has any role in the host defense and/or pathogenesis of clinically relevant A. baumannii infection is unknown. Using a sublethal mouse pneumonia model, we demonstrate that an extensively drug-resistant clinical isolate (ICII) of A. baumannii exhibits reduced/delayed early pulmonary neutrophil recruitment, higher lung persistence, and, most importantly, elicits enhanced IL-1ß/IL-18 production and lung damage through NLRP3 inflammasome, in comparison with A. baumannii-type strain. A. baumannii infection-induced IL-1ß/IL-18 production is entirely dependent on NLRP3-ASC-caspase-1/caspase-11 pathway. Using Nlrp3-/- mice infection models, we further show that while NLRP3 inflammasome pathway contributes to host defense against A. baumannii clinical isolate, it is dispensable for protection against A. baumannii-type strain. Our study reveals a novel differential role for NLRP3 inflammasome pathway in the immunity against clinically relevant A. baumannii infections, and highlights inflammasome pathway as a potential immunomodulatory target.

Inflammasome-dependent IL-1ß release depends upon membrane permeabilisation.

Interleukin-1ß (IL-1ß) is a critical regulator of the inflammatory response. IL-1ß is not secreted through the conventional ER-Golgi route of protein secretion, and to date its mechanism of release has been unknown. Crucially, its secretion depends upon the processing of a precursor form following the activation of the multimolecular inflammasome complex. Using a novel and reversible pharmacological inhibitor of the IL-1ß release process, in combination with biochemical, biophysical, and real-time single-cell confocal microscopy with macrophage cells expressing Venus-labelled IL-1ß, we have discovered that the secretion of IL-1ß after inflammasome activation requires membrane permeabilisation, and occurs in parallel with the death of the secreting cell. Thus, in macrophages the release of IL-1ß in response to inflammasome activation appears to be a secretory process independent of nonspecific leakage of proteins during cell death. The mechanism of membrane permeabilisation leading to IL-1ß release is distinct from the unconventional secretory mechanism employed by its structural homologues fibroblast growth factor 2 (FGF2) or IL-1α, a process that involves the formation of membrane pores but does not result in cell death. These discoveries reveal key processes at the initiation of an inflammatory response and deliver new insights into the mechanisms of protein release.

A unique role for p53 in the regulation of M2 macrophage polarization.

P53 is critically important in preventing oncogenesis but its role in inflammation in general and in the function of inflammatory macrophages in particular is not clear. Here, we show that bone marrow-derived macrophages exhibit endogenous p53 activity, which is increased when macrophages are polarized to the M2 (alternatively activated macrophage) subtype. This leads to reduced expression of M2 genes. Nutlin-3a, which destabilizes the p53/MDM2 (mouse double minute 2 homolog) complex, promotes p53 activation and further downregulates M2 gene expression. In contrast, increased expression of M2 genes was apparent in M2-polarized macrophages from p53-deficient and p53 mutant mice. Furthermore, we show, in mice, that p53 also regulates M2 polarization in peritoneal macrophages from interleukin-4-challenged animals and that nutlin-3a retards the development of tolerance to Escherichia coli lipopolysaccharide. P53 acts via transcriptional repression of expression of c-Myc (v-myc avian myelocytomatosis viral oncogene homolog) gene by directly associating with its promoter. These data establish a role for the p53/MDM2/c-MYC axis as a physiological 'brake' to the M2 polarization process. This work reveals a hitherto unknown role for p53 in macrophages, provides further insight into the complexities of macrophage plasticity and raises the possibility that p53-activating drugs, many of which are currently being trialled clinically, may have unforeseen effects on macrophage function.

Assessment of thymic output in common variable immunodeficiency patients by evaluation of T cell receptor excision circles.

Common variable immunodeficiency (CVID) is a heterogeneous syndrome characterized by repeated infections and hypogammaglobulinaemia. Additionally, T-cell abnormalities including lymphopenia, decreased proliferation to mitogens and antigens, and the reduced production and expression of cytokines, have also been observed. In this study we have investigated the expression of naive, memory and activation markers in T-cell subpopulations in 17 CVID patients in comparison to age-matched normal controls. The numbers of CD4+ T cells, including CD45RA+CD62L+ and, to a lesser extent, CD45RA-CD62L+/RA+CD62L- were significantly reduced in patients, whereas CD8+ T cells were within normal range. In contrast, HLA-DR+ cells were increased both in CD4+ and CD8+ T cells. To assess the thymic output, we analysed the presence of T-cell receptor excision circles (TRECs) in CD4+ and CD8+ T cells by quantitative PCR. TRECs were decreased significantly in patients and the rate of TREC loss was higher with increasing age. TRECs correlated with naive CD4+ T cells, whereas there was an inverse relationship between TRECs and CD8+HLA-DR+ and CD8+CD45RA-CD62L+/RA+CD62L- T cells. Our results suggest the presence of a defect in the naive T cell compartment with origin at the thymic level in CVID, and indicate that TREC may be a useful marker to monitor thymic function in this primary immunodeficiency.

New immunosuppressive drug PNU156804 blocks IL-2-dependent proliferation and NF-kappa B and AP-1 activation.

We had previously shown that the drug undecylprodigiosin (UP) blocks human lymphocyte proliferation in vitro. We have now investigated the mechanism of action of a new analogue of UP, PNU156804, which shows a more favorable activity profile than UP in mice. We demonstrate here that the biological effect of PNU156804 in vitro is indistinguishable from UP: PNU156804 blocks human T cell proliferation in mid-late G1, as determined by cell cycle analysis, expression of cyclins, and cyclin-dependent kinases and retinoblastoma phosphorylation. In addition, we show that PNU156804 does not block significantly the induction of either IL-2 or IL-2R alpha- and gamma-chains but inhibits IL-2-dependent T cell proliferation. We have investigated several molecular pathways that are known to be activated by IL-2 in T cells. We show that PNU156804 does not inhibit c-myc and bcl-2 mRNA induction. On the other hand, PNU156804 efficiently inhibits the activation of the NF-kappa B and AP-1 transcription factors. PNU156804 inhibition of NF-kappa B activation is due to the inhibition of the degradation of I kappa B-alpha and I kappa B-beta. PNU156804 action is restricted to some signaling pathways; it does not affect NF-kappa B activation by PMA in T cells but blocks that induced by CD40 cross-linking in B lymphocytes. We conclude that the prodigiosin family of immunosuppressants is a new family of molecules that show a novel target specificity clearly distinct from that of other immunosuppressive drugs such as cyclosporin A, FK506, and rapamycin.

Characterization of the new immunosuppressive drug undecylprodigiosin in human lymphocytes: retinoblastoma protein, cyclin-dependent kinase-2, and cyclin-dependent kinase-4 as molecular targets.

Undecylprodigiosin (UP) is the first described member of a family of related compounds showing immunosuppressive activity. We have investigated the biological effect and mechanism of action of UP in human lymphocytes. We show that UP blocks the proliferation of purified peripheral human T and B lymphocytes with an IC50 of 3 to 8 ng/ml and following stimulation by all mitogens used, with no effect on cell death. At the concentrations active on fresh lymphocytes, UP has no significant effect on the proliferation of different leukemic cell lines. UP blocks T cell activation in mid to late G1 phase and before entry into S phase, as shown by analysis of the cell cycle and of the expression of c-myc, IL-2, transferrin receptor, and B-myb. UP inhibits only partially the expression of IL-2R, suggesting that the major target of UP is localized downstream from the interaction between IL-2 and its receptor. The expression of cell cycle genes was investigated. The phosphorylation of the retinoblastoma protein was completely blocked by UP, an event alone sufficient to explain the block of S phase entry and the inhibition of proliferation. The induction of cyclin D2 and the decrease in p27 were not inhibited by UP, whereas the induction of cyclin E, cyclin A, cyclin-dependent kinase-2, and cyclin-dependent kinase-4 was strongly inhibited, potentially explaining the inhibition of retinoblastoma protein phosphorylation. These data clearly show that the site of action of UP is different from that of both cyclosporin A and rapamycin, and that this new class of compounds may, therefore, be good candidates for combined therapy.