Correction: The Influence of Programmed Cell Death in Myeloid Cells on Host Resilience to Infection with Legionella pneumophila or Streptococcus pyogenes.

[This corrects the article DOI: 10.1371/journal.ppat.1006032.].

The Influence of Programmed Cell Death in Myeloid Cells on Host Resilience to Infection with Legionella pneumophila or Streptococcus pyogenes.

Pathogen clearance and host resilience/tolerance to infection are both important factors in surviving an infection. Cells of the myeloid lineage play important roles in both of these processes. Neutrophils, monocytes, macrophages, and dendritic cells all have important roles in initiation of the immune response and clearance of bacterial pathogens. If these cells are not properly regulated they can result in excessive inflammation and immunopathology leading to decreased host resilience. Programmed cell death (PCD) is one possible mechanism that myeloid cells may use to prevent excessive inflammation. Myeloid cell subsets play roles in tissue repair, immune response resolution, and maintenance of homeostasis, so excessive PCD may also influence host resilience in this way. In addition, myeloid cell death is one mechanism used to control pathogen replication and dissemination. Many of these functions for PCD have been well defined in vitro, but the role in vivo is less well understood. We created a mouse that constitutively expresses the pro-survival B-cell lymphoma (bcl)-2 protein in myeloid cells (CD68(bcl2tg), thus decreasing PCD specifically in myeloid cells. Using this mouse model we explored the impact that decreased cell death of these cells has on infection with two different bacterial pathogens, Legionella pneumophila and Streptococcus pyogenes. Both of these pathogens target multiple cell death pathways in myeloid cells, and the expression of bcl2 resulted in decreased PCD after infection. We examined both pathogen clearance and host resilience and found that myeloid cell death was crucial for host resilience. Surprisingly, the decreased myeloid PCD had minimal impact on pathogen clearance. These data indicate that the most important role of PCD during infection with these bacteria is to minimize inflammation and increase host resilience, not to aid in the clearance or prevent the spread of the pathogen.

Pneumococcal neuraminidase activates TGF-ß signalling.

Neuraminidase A (NanA) is an important virulence factor that is anchored to the pneumococcal cell wall and cleaves sialic acid on host substrates. We noted that a secreted allele of NanA was over-represented in invasive pneumococcal isolates and promoted the development of meningitis when swapped into the genome of non-meningitis isolates replacing cell wall-anchored NanA. Both forms of recombinant NanA directly activated transforming growth factor (TGF)-ß, increased SMAD signalling and promoted loss of endothelial tight junction ZO-1. However, in assays using whole bacteria, only the cell-bound NanA decreased expression of ZO-1 and showed NanA dependence of bacterial invasion of endothelial cells. We conclude that NanA secretion versus retention on the cell surface does not influence neurotropism of clinical isolates. However, we describe a new NanA-TGF-ß signalling axis that leads to decreased blood-brain barrier integrity and enhances bacterial invasion.

Host-pathogen interactions in bacterial meningitis.

Bacterial meningitis is a devastating disease occurring worldwide with up to half of the survivors left with permanent neurological sequelae. Due to intrinsic properties of the meningeal pathogens and the host responses they induce, infection can cause relatively specific lesions and clinical syndromes that result from interference with the function of the affected nervous system tissue. Pathogenesis is based on complex host-pathogen interactions, some of which are specific for certain bacteria, whereas others are shared among different pathogens. In this review, we summarize the recent progress made in understanding the molecular and cellular events involved in these interactions. We focus on selected major pathogens, Streptococcus pneumonia, S. agalactiae (Group B Streptococcus), Neisseria meningitidis, and Escherichia coli K1, and also include a neglected zoonotic pathogen, Streptococcus suis. These neuroinvasive pathogens represent common themes of host-pathogen interactions, such as colonization and invasion of mucosal barriers, survival in the blood stream, entry into the central nervous system by translocation of the blood-brain and blood-cerebrospinal fluid barrier, and induction of meningeal inflammation, affecting pia mater, the arachnoid and subarachnoid spaces.

Type I interferon production induced by Streptococcus pyogenes-derived nucleic acids is required for host protection.

Streptococcus pyogenes is a Gram-positive human pathogen that is recognized by yet unknown pattern recognition receptors (PRRs). Engagement of these receptor molecules during infection with S. pyogenes, a largely extracellular bacterium with limited capacity for intracellular survival, causes innate immune cells to produce inflammatory mediators such as TNF, but also type I interferon (IFN). Here we show that signaling elicited by type I IFNs is required for successful defense of mice against lethal subcutaneous cellulitis caused by S. pyogenes. Type I IFN signaling was accompanied with reduced neutrophil recruitment to the site of infection. Mechanistic analysis revealed that macrophages and conventional dendritic cells (cDCs) employ different signaling pathways leading to IFN-beta production. Macrophages required IRF3, STING, TBK1 and partially MyD88, whereas in cDCs the IFN-beta production was fully dependent on IRF5 and MyD88. Furthermore, IFN-beta production by macrophages was dependent on the endosomal delivery of streptococcal DNA, while in cDCs streptococcal RNA was identified as the IFN-beta inducer. Despite a role of MyD88 in both cell types, the known IFN-inducing TLRs were individually not required for generation of the IFN-beta response. These results demonstrate that the innate immune system employs several strategies to efficiently recognize S. pyogenes, a pathogenic bacterium that succeeded in avoiding recognition by the standard arsenal of TLRs.

Autophagy supports Candida glabrata survival during phagocytosis.

The opportunistic human fungal pathogen Candida glabrata is confronted with phagocytic cells of the host defence system. Survival of internalized cells is thought to contribute to successful dissemination. We investigated the reaction of engulfed C. glabrata cells using fluorescent protein fusions of the transcription factors CgYap1 and CgMig1 and catalase CgCta1. The expression level and peroxisomal localization of catalase was used to monitor the metabolic and stress status of internalized C. glabrata cells. These reporters revealed that the phagocytosed C. glabrata cells were exposed to transient oxidative stress and starved for carbon source. Cells trapped within macrophages increased their peroxisome numbers indicating a metabolic switch. Prolonged phagocytosis caused a pexophagy-mediated decline in peroxisome numbers. Autophagy, and in particular pexophagy, contributed to survival of C. glabrata during engulfment. Mutants lacking CgATG11 or CgATG17, genes required for pexophagy and non-selective autophagy, respectively, displayed reduced survival rates. Furthermore, both CgAtg11 and CgAtg17 contribute to survival, since the double mutant was highly sensitive to engulfment. Inhibition of peroxisome formation by deletion of CgPEX3 partially restored viability of CgATG11 deletion mutants during engulfment. This suggests that peroxisome formation and maintenance might sequester resources required for optimal survival. Mobilization of intracellular resources via autophagy is an important virulence factor that supports the viability of C. glabrata in the phagosomal compartment of infected innate immune cells.

Tristetraprolin is required for full anti-inflammatory response of murine macrophages to IL-10.

IL-10 is essential for inhibiting chronic and acute inflammation by decreasing the amounts of proinflammatory cytokines made by activated macrophages. IL-10 controls proinflammatory cytokine and chemokine production indirectly via the transcription factor Stat3. One of the most physiologically significant IL-10 targets is TNF-alpha, a potent proinflammatory mediator that is the target for multiple anti-TNF-alpha clinical strategies in Crohn's disease and rheumatoid arthritis. The anti-inflammatory effects of IL-10 seem to be mediated by several incompletely understood transcriptional and posttranscriptional mechanisms. In this study, we show that in LPS-activated bone marrow-derived murine macrophages, IL-10 reduces the mRNA and protein levels of TNF-alpha and IL-1alpha in part through the RNA destabilizing factor tristetraprolin (TTP). TTP is known for its central role in destabilizing mRNA molecules containing class II AU-rich elements in 3' untranslated regions. We found that IL-10 initiates a Stat3-dependent increase of TTP expression accompanied by a delayed decrease of p38 MAPK activity. The reduction of p38 MAPK activity releases TTP from the p38 MAPK-mediated inhibition, thereby resulting in diminished mRNA and protein levels of proinflammatory cytokines. These findings establish that TTP is required for full responses of bone marrow-derived murine macrophages to IL-10.

The RNA-binding protein tristetraprolin schedules apoptosis of pathogen-engaged neutrophils during bacterial infection.

Protective responses against pathogens require a rapid mobilization of resting neutrophils and the timely removal of activated ones. Neutrophils are exceptionally short-lived leukocytes, yet it remains unclear whether the lifespan of pathogen-engaged neutrophils is regulated differently from that in the circulating steady-state pool. Here, we have found that under homeostatic conditions, the mRNA-destabilizing protein tristetraprolin (TTP) regulates apoptosis and the numbers of activated infiltrating murine neutrophils but not neutrophil cellularity. Activated TTP-deficient neutrophils exhibited decreased apoptosis and enhanced accumulation at the infection site. In the context of myeloid-specific deletion of Ttp, the potentiation of neutrophil deployment protected mice against lethal soft tissue infection with Streptococcus pyogenes and prevented bacterial dissemination. Neutrophil transcriptome analysis revealed that decreased apoptosis of TTP-deficient neutrophils was specifically associated with elevated expression of myeloid cell leukemia 1 (Mcl1) but not other antiapoptotic B cell leukemia/lymphoma 2 (Bcl2) family members. Higher Mcl1 expression resulted from stabilization of Mcl1 mRNA in the absence of TTP. The low apoptosis rate of infiltrating TTP-deficient neutrophils was comparable to that of transgenic Mcl1-overexpressing neutrophils. Our study demonstrates that posttranscriptional gene regulation by TTP schedules the termination of the antimicrobial engagement of neutrophils. The balancing role of TTP comes at the cost of an increased risk of bacterial infections.

Tristetraprolin Limits Inflammatory Cytokine Production in Tumor-Associated Macrophages in an mRNA Decay-Independent Manner.

Tristetraprolin (TTP) is an inducible zinc finger AU-rich RNA-binding protein essential for enforcing degradation of mRNAs encoding inflammatory chemokines and cytokines. Most studies on TTP center on the connection between mRNA half-life and inflammatory output, because loss of TTP amplifies inflammation by increasing the stability of AU-rich mRNAs. Here, we focused on how TTP controls cytokine and chemokine production in the nonresolving inflammation of cancer using tissue-specific approaches. In contrast with model in vitro macrophage systems, we found constitutive TTP expression in late-stage tumor-associated macrophages (TAM). However, TTP's effects on AU-rich mRNA stability were negligible and limited by constitutive p38α MAPK activity, which was the main driver of proinflammatory cytokine production in TAMs at the posttranscriptional level. Instead, elimination of TTP caused excessive protein production of inflammatory mediators, suggesting TTP-dependent translational suppression of AU-rich mRNAs. Manipulation of the p38α-TTP axis in macrophages has significant effects on the growth of tumors and therefore represents a means to manipulate inflammation in the tumor microenvironment.

Innate immune response to Streptococcus pyogenes depends on the combined activation of TLR13 and TLR2.

Innate immune recognition of the major human-specific Gram-positive pathogen Streptococcus pyogenes is not understood. Here we show that mice employ Toll-like receptor (TLR) 2- and TLR13-mediated recognition of S. pyogenes. These TLR pathways are non-redundant in the in vivo context of animal infection, but are largely redundant in vitro, as only inactivation of both of them abolishes inflammatory cytokine production by macrophages and dendritic cells infected with S. pyogenes. Mechanistically, S. pyogenes is initially recognized in a phagocytosis-independent manner by TLR2 and subsequently by TLR13 upon internalization. We show that the TLR13 response is specifically triggered by S. pyogenes rRNA and that Tlr13-/- cells respond to S. pyogenes infection solely by engagement of TLR2. TLR13 is absent from humans and, remarkably, we find no equivalent route for S. pyogenes RNA recognition in human macrophages. Phylogenetic analysis reveals that TLR13 occurs in all kingdoms but only in few mammals, including mice and rats, which are naturally resistant against S. pyogenes. Our study establishes that the dissimilar expression of TLR13 in mice and humans has functional consequences for recognition of S. pyogenes in these organisms.

Regulation of Candida glabrata oxidative stress resistance is adapted to host environment.

The human fungal pathogen Candida glabrata is related to Saccharomyces cerevisiae but has developed high resistance against reactive oxygen species. We find that induction of conserved genes encoding antioxidant functions is dependent on the transcription factors CgYap1 and CgSkn7 which cooperate for promoter recognition. Superoxide stress resistance of C. glabrata is provided by superoxide dismutase CgSod1, which is not dependent on CgYap1/Skn7. Only double mutants lacking both CgSod1 and CgYap1 were efficiently killed by primary mouse macrophages. Our results suggest that in C. glabrata the regulation of key genes providing stress protection is adopted to meet a host-pathogen situation.

Group A streptococcus activates type I interferon production and MyD88-dependent signaling without involvement of TLR2, TLR4, and TLR9.

Bacterial pathogens are recognized by the innate immune system through pattern recognition receptors, such as Toll-like receptors (TLRs). Engagement of TLRs triggers signaling cascades that launch innate immune responses. Activation of MAPKs and NF-kappaB, elements of the major signaling pathways induced by TLRs, depends in most cases on the adaptor molecule MyD88. In addition, Gram-negative or intracellular bacteria elicit MyD88-independent signaling that results in production of type I interferon (IFN). Here we show that in mouse macrophages, the activation of MyD88-dependent signaling by the extracellular Gram-positive human pathogen group A streptococcus (GAS; Streptococcus pyogenes) does not require TLR2, a receptor implicated in sensing of Gram-positive bacteria, or TLR4 and TLR9. Redundant engagement of either of these TLR molecules was excluded by using TLR2/4/9 triple-deficient macrophages. We further demonstrate that infection of macrophages by GAS causes IRF3 (interferon-regulatory factor 3)-dependent, MyD88-independent production of IFN. Surprisingly, IFN is induced also by GAS lacking slo and sagA, the genes encoding cytolysins that were shown to be required for IFN production in response to other Gram-positive bacteria. Our data indicate that (i) GAS is recognized by a MyD88-dependent receptor other than any of those typically used by bacteria, and (ii) GAS as well as GAS mutants lacking cytolysin genes induce type I IFN production by similar mechanisms as bacteria requiring cytoplasmic escape and the function of cytolysins.