Macrophages infected with Mycobacterium tuberculosis undergo increased rates of apoptosis. Important objectives are to define the microbial factors that cause apoptosis, the mechanisms involved and the impact on infection. The 19-kDa M. tuberculosis glycolipoprotein (p19) is both cell wall-associated and secreted and is a candidate virulence factor. We investigated the potential of recombinant, His-tagged p19 lacking the secretion/acylation signal to induce macrophage apoptosis. The TUNEL assay and annexin V binding to membrane phosphatidylserine were used to measure apoptosis. The results show that p19 does act to induce apoptosis in differentiated THP-1 cells and monocyte-derived macrophages and that this effect is both dose- and time-dependent. Furthermore, this effect of p19 is Toll-like receptor (TLR)-2-mediated because preincubation of either THP-1 cells or TLR-2-expressing CHO cells with anti-TLR-2 mAb inhibited apoptosis induced by p19. Apoptosis of macrophages in response to p19 was found to be caspase-8 dependent and caspase-9 independent consistent with a transmembrane pathway signaling cell death through TLR-2. The viability of M. tuberculosis in cells undergoing apoptosis induced by p19 was significantly reduced suggesting the possibility that this may favor containment of infection. Although native p19 is a mycobacterial glycolipoprotein, based upon the use of recombinant p19 where the acylation signal had been removed, we conclude that it is the polypeptide component of p19 that is responsible for signaling through TLR-2 and that the lipid moiety is not required.

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The human leishmaniasis are persistent infections of macrophages caused by protozoa of the genus Leishmania. The chronic nature of these infections is in part related to induction of macrophage deactivation, linked to activation of the Src homology 2 domain containing tyrosine phosphatase-1 (SHP-1) in infected cells. To investigate the mechanism of SHP-1 activation, lysates of Leishmania donovani promastigotes were subjected to SHP-1 affinity chromatography and proteins bound to the matrix were sequenced by mass spectrometry. This resulted in the identification of Leishmania elongation factor-1␣ (EF-1␣) as a SHP-1-binding protein. Purified Leishmania EF-1␣, but not host cell EF-1␣, bound directly to SHP-1 in vitro leading to its activation. Three independent lines of evidence indicated that Leishmania EF-1␣ may be exported from the phagosome thereby enabling targeting of host SHP-1. First, cytosolic fractions prepared from macrophages infected with [35 S]methionine-labeled organisms contained Leishmania EF-1␣. Second, confocal, fluorescence microscopy using Leishmania-specific antisera detected Leishmania EF-1␣ in the cytosol of infected cells. Third, co-immunoprecipitation showed that Leishmania EF-1␣ was associated with SHP-1 in vivo in infected cells. Finally, introduction of purified Leishmania EF-1␣, but not the corresponding host protein into macrophages activated SHP-1 and blocked the induction of inducible nitricoxide synthase expression in response to interferon-␥. Thus, Leishmania EF-1␣ is identified as a novel SHP-1-binding and activating protein that recapitulates the deactivated phenotype of infected macrophages.According to the latest WHO report, 12 million people are affected by leishmaniasis worldwide and 2 million new cases occur each year (Leishmaniases Control, www.who.int/ health-topics/leishmaniasis.htm, updated 2000). Moreover, the incidence of the leishmaniasis has been on the rise because of multiple factors including the AIDS epidemic, increased international travel, lack of effective vaccines, difficulty in controlling vectors, international conflicts, and the development of resistance to chemotherapy. Progress in controlling the leishmaniasis will require improved understanding of pathogenesis to identify novel drug targets or vaccine candidates.Leishmania donovani is the major causative agent of human visceral leishmaniasis. Leishmania live as either extracellular, flagellated promastigotes in the digestive tracts of their sand fly vectors or as nonflagellated amastigotes within macrophages, where they survive and replicate within phagolysosomes. Macrophages as part of both the innate and acquired immune systems are programmed to ingest and destroy intracellular pathogens. Hence, the mechanisms used by Leishmania and other intracellular pathogens to evade elimination by macrophages are important issues in cell biology and immunology. Infected macrophages are often refractory to cell activation (1, 2) and recent evidence suggests that this is related to impaired cell signaling (1-4) br...

Summary Lymphotoxin β-receptor (LTβR)-signalling orchestrates lymphoid neogenesis and subsequent tertiary lymphoid structures (TLS) 1 , 2 , associated with severe chronic inflammatory diseases spanning multiple organ systems 3 – 6 . How LTβR-signalling drives chronic tissue damage particularly in the lung, which mechanism(s) regulate this process, and whether LTβR-blockade might be of therapeutic value has remained unclear. Here we demonstrate increased expression of LTβR-ligands on adaptive and innate immune-cells, enhanced non-canonical NF-κB signalling and enriched LTβR-target gene expression in epithelial cells of lungs from patients with smoking-associated chronic obstructive pulmonary disease (COPD) and mice exposed to chronic cigarette smoke. Therapeutic inhibition of LTβR-signalling in young and aged mice disrupted smoking-related inducible bronchus-associated lymphoid tissue (iBALT), induced lung tissue regeneration, and reverted airway-fibrosis and systemic muscle wasting. Mechanistically, LTβR-signalling blockade dampened epithelial non-canonical NF-κB activation, reduced TGFβ-signalling in airways, induced regeneration by preventing epithelial cell-death and by activating Wnt/β-catenin-signalling in alveolar epithelial progenitor cells. These findings highlight that LTβR-signalling inhibition represents a viable therapeutic option combining anti-TLS, anti-apoptotic with tissue regenerative strategies.

Leishmania disease expression has been linked to IL-10. In this study, we investigated the regulation of IL-10 production by macrophages infected with Leishmania donovani. Infection of either murine or human macrophages brought about selective phosphorylation of Akt-2 in a PI3K-dependent manner. These events were linked to phosphorylation and inactivation of glycogen synthase kinase-3β (GSK-3β) at serine 9, as the latter was abrogated by inhibition of either PI3K or Akt. One of the transcription factors that is negatively regulated by GSK-3β is CREB, which itself positively regulates IL-10 expression. Infection of macrophages with leishmania induced phosphorylation of CREB at serine 133, and this was associated with enhanced CREB DNA binding activity and induction of IL-10. Similar to phosphorylation of GSK-3β, both phosphorylation of CREB at serine 133 and CREB DNA binding activity were abrogated in cells treated with inhibitors of either PI3K or Akt prior to infection. Furthermore, disruption of this pathway either by inhibition of Akt or by overexpression of GSK-3β markedly attenuated IL-10 production in response to leishmania. Thus, GSK-3β negatively regulates myeloid cell IL-10 production in response to leishmania. Switching off GSK-3β promotes disease pathogenesis.