Heme amplifies the innate immune response to microbial molecules through spleen tyrosine kinase (Syk)-dependent reactive oxygen species generation.

Infectious diseases that cause hemolysis are among the most threatening human diseases, because of severity and/or global distribution. In these conditions, hemeproteins and heme are released, but whether heme affects the inflammatory response to microorganism molecules remains to be characterized. Here, we show that heme increased the lethality and cytokine secretion induced by LPS in vivo and enhanced the secretion of cytokines by macrophages stimulated with various agonists of innate immune receptors. Activation of nuclear factor κB (NF-κB) and MAPKs and the generation of reactive oxygen species were essential to the increase in cytokine production induced by heme plus LPS. This synergistic effect of heme and LPS was blocked by a selective inhibitor of spleen tyrosine kinase (Syk) and was abrogated in dendritic cells deficient in Syk. Moreover, inhibition of Syk and the downstream molecules PKC and PI3K reduced the reactive oxygen species generation by heme. Our results highlight a mechanism by which heme amplifies the secretion of cytokines triggered by microbial molecule activation and indicates possible pathways for therapeutic intervention during hemolytic infectious diseases.

Novel role for the double-stranded RNA-activated protein kinase PKR: modulation of macrophage infection by the protozoan parasite Leishmania.

The evolution of Leishmania infection depends on the balance between microbicidal and suppressor macrophage functions. Double-stranded RNA (dsRNA)-activated protein kinase R (PKR), a classic antiviral protein, is able to regulate a number of signaling pathways and macrophage functions. We investigated the possible role of PKR in the modulation of Leishmania infection. Our data demonstrated that Leishmania amazonensis infection led to PKR activation and increased PKR levels. Consistently, in macrophages from PKR knockout 129Sv/Ev mice and RAW-264.7 cells stably expressing a dominant-negative (DN) construct of PKR (DN-PKR), L. amazonensis infection was strongly reduced. The treatment of infected macrophages with the synthetic double-stranded RNA poly(I:C), a potent PKR inductor, increased L. amazonensis intracellular proliferation. This effect was reversed by 2-aminopurine (2-AP), a pharmacological inhibitor of PKR, as well as by the expression of DN-PKR. NO release induced by dsRNA treatment was inhibited by L. amazonensis through NF-kappaB modulation. PKR activation induced by dsRNA also resulted in IL-10 production, whose neutralization with specific antibody completely abrogated L. amazonensis proliferation. Our data demonstrated a new role of PKR in protozoan parasitic infection through IL-10 modulation.

IL-27 enhances Leishmania amazonensis infection via ds-RNA dependent kinase (PKR) and IL-10 signaling.

The protozoan parasite Leishmania infects and replicates in macrophages, causing a spectrum of diseases in the human host, varying from cutaneous to visceral clinical forms. It is known that cytokines modulate the immunological response against Leishmania and are relevant for infection resolution. Here, we report that Interleukin (IL)-27 increases Leishmania amazonensis replication in human and murine macrophages and that the blockage of the IL-10 receptor on the surface of infected cells abolished the IL-27-mediated enhancement of Leishmania growth. IL-27 induced the activation/phosphorylation of protein kinase R (PKR) in macrophages, and PKR blockage or PKR gene deletion abrogated the enhancement of the parasite growth driven by IL-27, as well as the L. amazonensis-induced macrophage production of IL-27. We also observed that L. amazonensis-induced expression of IL-27 depends on type I interferon signaling and the engagement of Toll-like receptor 2. Treatment of Leishmania-infected mice with IL-27 increased lesion size and parasite loads in the footpad and lymph nodes of infected animals, indicating that this cytokine exerts a local and a systemic effect on parasite growth and propagation. In conclusion, we show that IL-27 is a L. amazonensis-enhancing factor and that the PKR/IFN1 axis and IL-10 are critical mediators of this IL-27 induced effect.

NF-kappaB-mediated repression of iNOS expression in Leishmania amazonensis macrophage infection.

Host invasion by pathogens is frequently associated with the activation of nuclear factor kappaB (NF-kappaB), which modulates the expression of genes involved in the immunological response of the host. However, pathogens may also subvert these mechanisms to secure their survival. We describe the effect of Leishmania amazonensis infection on NF-kappaB transcriptional factor activation in macrophages and the subsequent reduction in inducible nitric oxide synthase (iNOS) expression. L. amazonensis promastigote infection activates the p50/p50 NF-kappaB complex, a classic transcriptional repressor. Interestingly, L. amazonensis promotes the change of the classical p65/p50 NF-kappaB dimer induced by LPS, leading to the p50/p50 NF-kappaB complex activation in macrophages stimulated with LPS. Moreover, this parasite promotes the reduction of p65 total levels in infected macrophages. All these effects contribute to the observation that this parasite is able to restrain the NF-kappaB-dependent transcriptional activity induced by LPS. Strikingly, L. amazonensis reduces the mRNA levels of the iNOS in addition to protein expression and the production of nitric oxide in LPS-stimulated macrophages. Accordingly, as revealed by reporter-gene assays, L. amazonensis-induced iNOS repression requires NF-kappaB sites in the iNOS promoter region. In summary, our results suggest that L. amazonensis has developed an adaptive strategy to escape from host defense by activating the NF-kappaB repressor complex p50/p50. The activation of this specific host transcriptional response negatively regulates the expression of iNOS, favoring the establishment and success of L. amazonensis infection.

Mycobacterium leprae induces NF-kappaB-dependent transcription repression in human Schwann cells.

Mycobacterium leprae, the causative agent of leprosy, invades peripheral nerve Schwann cells, resulting in deformities associated with this disease. NF-kappaB is an important transcription factor involved in the regulation of host immune antimicrobial responses. We aimed in this work to investigate NF-kappaB signaling pathways in the human ST88-14 Schwannoma cell line infected with M. leprae. Gel shift and supershift assays indicate that two NF-kappaB dimers, p65/p50 and p50/p50, translocate to the nucleus in Schwann cells treated with lethally irradiated M. leprae. Consistent with p65/p50 and p50/p50 activation, we observed IkappaB-alpha degradation and reduction of p105 levels. The nuclear translocation of p50/p50 complex due to M. leprae treatment correlated with repression of NF-kappaB-driven transcription induced by TNF-alpha. Moreover, thalidomide inhibited p50 homodimer nuclear translocation induced by M. leprae and consequently rescues Schwann cells from NF-kappaB-dependent transcriptional repression. Here, we report for the first time that M. leprae induces NF-kappaB activation in Schwann cells and thalidomide is able to modulate this activation.

Caspase-8 activity prevents type 2 cytokine responses and is required for protective T cell-mediated immunity against Trypanosoma cruzi infection.

During Trypanosoma cruzi infection, T cells up-regulate caspase-8 activity. To assess the role of caspase-8 in T cell-mediated immunity, we investigated the effects of caspase-8 inhibition on T cells in viral FLIP (v-FLIP) transgenic mice. Compared with wild-type controls, increased parasitemia was observed in v-FLIP mice infected with T. cruzi. There was a profound decrease in expansion of both CD4 and CD8 T cell subsets in the spleens of infected v-FLIP mice. We did not find differences in activation ratios of T cells from transgenic or wild-type infected mice. However, the numbers of memory/activated CD4 and CD8 T cells were markedly reduced in v-FLIP mice, possibly due to defective survival. We also found decreased production of IL-2 and increased secretion of type 2 cytokines, IL-4 and IL-10, which could enhance susceptibility to infection. Similar, but less pronounced, alterations were observed in mice treated with the caspase-8 inhibitor, zIETD. Furthermore, blockade of caspase-8 by zIETD in vitro mimicked the effects observed on T. cruzi infection in vivo, affecting the generation of activated/memory T cells and T cell cytokine production. Caspase-8 is also required for NF-kappaB signaling upon T cell activation. Blockade of caspase-8 by either v-FLIP expression or treatment with zIETD peptide decreased NF-kappaB responses to TCR:CD3 engagement in T cell cultures. These results suggest a critical role for caspase-8 in the establishment of T cell memory, cell signaling, and regulation of cytokine responses during protozoan infection.