Intracellular C3 protects ß-cells from IL-1ß-driven cytotoxicity via interaction with Fyn-related kinase.

One of the hallmarks of type 1 but also type 2 diabetes is pancreatic islet inflammation, associated with altered pancreatic islet function and structure, if unresolved. IL-1ß is a proinflammatory cytokine which detrimentally affects ß-cell function. In the course of diabetes, complement components, including the central complement protein C3, are deregulated. Previously, we reported high C3 expression in human pancreatic islets, with upregulation after IL-1ß treatment. In the current investigation, using primary human and rodent material and CRISPR/Cas9 gene-edited ß-cells deficient in C3, or producing only cytosolic C3 from a noncanonical in-frame start codon, we report a protective effect of C3 against IL-1ß-induced ß-cell death, that is attributed to the cytosolic fraction of C3. Further investigation revealed that intracellular C3 alleviates IL-1ß-induced ß-cell death, by interaction with and inhibition of Fyn-related kinase (FRK), which is involved in the response of ß-cells to cytokines. Furthermore, these data were supported by increased ß-cell death in vivo in a ß-cell-specific C3 knockout mouse. Our data indicate that a functional, cytoprotective association exists between FRK and cytosolic C3.

Alternative translation and retrotranslocation of cytosolic C3 that detects cytoinvasive bacteria.

Complement C3 was originally regarded as a serum effector protein, although recent data has emerged suggesting that intracellular C3 can also regulate basic cellular processes. Despite the growing interest in intracellular C3 functions, the mechanism behind its generation has not been demonstrated. In this study we show that C3 can be expressed from an alternative translational start site, resulting in C3 lacking the signal peptide, which is therefore translated in the cytosol. In contrast to the secreted form, alternatively translated cytosolic C3 is not glycosylated, is present mainly in a reduced state, and is turned over by the ubiquitin-proteasome system. C3 can also be retrotranslocated from the endoplasmic reticulum into the cytosol, structurally resembling secreted C3. Finally, we demonstrate that intracellular cytosolic C3 can opsonize invasive Staphylococcus aureus within epithelial cell, slowing vacuolar escape as well as impacting bacterial survival on subsequent exposure to phagocytes. Our work therefore reveals the existence and origin of intracellular, cytosolic C3, and demonstrates functions for cytosolic C3 in intracellular detection of cytoinvasive pathogens.

c-Myc is a novel Leishmania virulence factor by proxy that targets the host miRNA system and is essential for survival in human macrophages.

Leishmania species are intracellular protozoan pathogens that have evolved to successfully infect and deactivate host macrophages. How this deactivation is brought about is not completely understood. Recently, microRNAs (miRNAs) have emerged as ubiquitous regulators of macrophage gene expression that contribute to shaping the immune responses to intracellular pathogens. Conversely, several pathogens have evolved the ability to exploit host miRNA expression to manipulate host-cell phenotype. However, very little is known about the mechanisms used by intracellular pathogens to drive changes in host-cell miRNA abundance. Using miRNA expression profiling of Leishmania donovani-infected human macrophages, we show here that Leishmania infection induced a genome-wide down-regulation of host miRNAs. This repression occurred at the level of miRNA gene transcription, because the synthesis rates of primary miRNAs were significantly decreased in infected cells. miRNA repression depended on the host macrophage transcription factor c-Myc. Indeed, the expression of host c-Myc was markedly up-regulated by Leishmania infection, and c-Myc silencing reversed the miRNA suppression. Furthermore, c-Myc silencing significantly reduced intracellular survival of Leishmania, demonstrating that c-Myc is essential for Leishmania pathogenesis. Taken together, these findings identify c-Myc not only as being responsible for miRNA repression in Leishmania-infected macrophages but also as a novel and essential virulence factor by proxy that promotes Leishmania survival.

Leishmania donovani chaperonin 10 regulates parasite internalization and intracellular survival in human macrophages.

Protozoa of the genus Leishmania infect macrophages in their mammalian hosts causing a spectrum of diseases known as the leishmaniases. The search for leishmania effectors that support macrophage infection is a focus of significant interest. One such candidate is leishmania chaperonin 10 (CPN10) which is secreted in exosomes and may have immunosuppressive properties. Here, we report for the first time that leishmania CPN10 localizes to the cytosol of infected macrophages. Next, we generated two genetically modified strains of Leishmania donovani (Ld): one strain overexpressing CPN10 (CPN10+++) and the second, a CPN10 single allele knockdown (CPN10+/-), as the null mutant was lethal. When compared with the wild-type (WT) parental strain, CPN10+/- Ld showed higher infection rates and parasite loads in human macrophages after 24 h of infection. Conversely, CPN10+++ Ld was associated with lower initial infection rates. This unexpected apparent gain-of-function for the knockdown could have been explained either by enhanced parasite internalization or by enhanced intracellular survival. Paradoxically, we found that CPN10+/- leishmania were more readily internalized than WT Ld, but also displayed significantly impaired intracellular survival. This suggests that leishmania CPN10 negatively regulates the rate of parasite uptake by macrophages while being required for intracellular survival. Finally, quantitative proteomics identified an array of leishmania proteins whose expression was positively regulated by CPN10. In contrast, many macrophage proteins involved in innate immunity were negatively regulated by CPN10. Taken together, these findings identify leishmania CPN10 as a novel effector with broad based effects on macrophage cell regulation and parasite survival.

Phenotypic Characterization of a Leishmania donovani Cyclophilin 40 Null Mutant.

Protozoan parasites of the genus Leishmania adapt to their arthropod and vertebrate hosts through the development of defined life cycle stages. Stage differentiation is triggered by environmental stress factors and has been linked to parasite chaperone activities. Using a null mutant approach we previously revealed important, nonredundant functions of the cochaperone cyclophilin 40 in L. donovani-infected macrophages. Here, we characterized in more detail the virulence defect of cyp40-/- null mutants. In vitro viability assays, infection tests using macrophages, and mixed infection experiments ruled out a defect of cyp40-/- parasites in resistance to oxidative and hydrolytic stresses encountered inside the host cell phagolysosome. Investigation of the CyP40-dependent proteome by quantitative 2D-DiGE analysis revealed up regulation of various stress proteins in the null mutant, presumably a response to compensate for the lack of CyP40. Applying transmission electron microscopy we showed accumulation of vesicular structures in the flagellar pocket of cyp40-/- parasites that we related to a significant increase in exosome production, a phenomenon previously linked to the parasite stress response. Together these data suggest that cyp40-/- parasites experience important intrinsic homeostatic stress that likely abrogates parasite viability during intracellular infection.

Outside in: Roles of complement in autophagy.

The complement system is a well-characterized cascade of extracellular serum proteins that is activated by pathogens and unwanted waste material. Products of activated complement signal to the host cells via cell surface receptors, eliciting responses such as removal of the stimulus by phagocytosis. The complement system therefore functions as a warning system, resulting in removal of unwanted material. This review describes how extracellular activation of the complement system can also trigger autophagic responses within cells, up-regulating protective homeostatic autophagy in response to perceived stress, but also initiating targeted anti-microbial autophagy in order to kill intracellular cytoinvasive pathogens. In particular, we will focus on recent discoveries that indicate that complement may also have roles in detection and autophagy-mediated disposal of unwanted materials within the intracellular environment. We therefore summarize the current evidence for complement involvement in autophagy, both by transducing signals across the cell membrane, as well as roles within the cellular environment. LINKED ARTICLES: This article is part of a themed issue on Canonical and non-canonical functions of the complement system in health and disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.14/issuetoc.

Interaction of Streptococcus pyogenes with extracellular matrix components resulting in immunomodulation and bacterial eradication.

Streptococcus pyogenes is a major human pathogen that causes a variety of diseases ranging from mild skin and throat infections to fatal septicemia. In severe invasive infections, S. pyogenes encounters and interacts with components of the extracellular matrix (ECM), including small leucine rich-proteoglycans (SLRPs). In this study, we report a novel antimicrobial role played by SLRPs biglycan, decorin, fibromodulin and osteoadherin, specifically in promoting the eradication of S. pyogenes in a human sepsis model of infection. SLRPs can be released from the ECM and de novo synthesized by a number of cell types. We reveal that infection of human monocytes by S. pyogenes induces the expression of decorin. Furthermore, we show that the majority of genetically distinct and clinically relevant S. pyogenes isolates interact with SLRPs resulting in decreased survival in blood killing assays. Biglycan and decorin induce TLR2 and TLR4 signaling cascades resulting in secretion of proinflammatory and chemotactic molecules and recruitment of professional phagocytes. Surprisingly, SLRP-mediated elimination of S. pyogenes occurs independently of TLR activation. Our results indicate that SLRPs act in concert with human serum, enhancing deposition of complement activation fragments and the classical activator C1q on the bacterial surface, facilitating efficient microbial eradication. Addition of the complement C3 inhibitor compstatin significantly reverses SLRP-induced blood killing, confirming active complement as a key mediator in SLRP-mediated bacterial destruction. Taken together our results add to the functional repertoire of SLRPs, expanding to encompass their role in controlling bacterial infection.

Antibacterial Fusion Proteins Enhance Moraxella catarrhalis Killing.

Moraxella catarrhalis is a human-specific commensal of the respiratory tract and an opportunistic pathogen. It is one of the leading cause of otitis media in children and of acute exacerbations in patients with chronic obstructive pulmonary disease, resulting in significant morbidity and economic burden. Vaccines and new immunotherapeutic strategies to treat this emerging pathogen are needed. Complement is a key component of innate immunity that mediates the detection, response, and subsequent elimination of invading pathogens. Many pathogens including M. catarrhalis have evolved complement evasion mechanisms, which include the binding of human complement inhibitors such as C4b-binding protein (C4BP) and Factor H (FH). Inhibiting C4BP and FH acquisition by M. catarrhalis may provide a novel therapeutic avenue to treat infections. To achieve this, we created two chimeric proteins that combined the Moraxella-binding domains of C4BP and FH fused to human immunoglobulin Fcs: C4BP domains 1 and 2 and FH domains 6 and 7 fused to IgM and IgG Fc, respectively. As expected, FH6-7/IgG displaced FH from the bacterial surface while simultaneously activating complement via Fc-C1q interactions, together increasing pathogen elimination. C4BP1-2/IgM also increased serum killing of the bacteria through enhanced complement deposition, but did not displace C4BP from the surface of M. catarrhalis. These Fc fusion proteins could act as anti-infective immunotherapies. Many microbes bind the complement inhibitors C4BP and FH through the same domains as M. catarrhalis, therefore these Fc fusion proteins may be promising candidates as adjunctive therapy against many different drug-resistant pathogens.

HIV-Infected Spleens Present Altered Follicular Helper T Cell (Tfh) Subsets and Skewed B Cell Maturation.

Follicular helper T (Tfh) cells within secondary lymphoid organs control multiple steps of B cell maturation and antibody (Ab) production. HIV-1 infection is associated with an altered B cell differentiation and Tfh isolated from lymph nodes of HIV-infected (HIV+) individuals provide inadequate B cell help in vitro. However, the mechanisms underlying this impairment of Tfh function are not fully defined. Using a unique collection of splenocytes, we compared the frequency, phenotype and transcriptome of Tfh subsets in spleens from HIV negative (HIV-) and HIV+ subjects. We observed an increase of CXCR5+PD-1highCD57-Tfh and germinal center (GC) CD57+ Tfh in HIV+ spleens. Both subsets showed a reduced mRNA expression of the transcription factor STAT-3, co-stimulatory, regulatory and signal transduction molecules as compared to HIV- spleens. Similarly, Foxp3 expressing follicular regulatory T (Tfr) cells were increased, suggesting sustained GC reactions in chronically HIV+ spleens. As a consequence, GC B cell populations were expanded, however, complete maturation into memory B cells was reduced in HIV+ spleens where we evidenced a compromised production of B cell-activating cytokines such as IL-4 and IL-10. Collectively our data indicate that, although Tfh proliferation and GC reactions seem to be ongoing in HIV-infected spleens, Tfh "differentiation" and expression of costimulatory molecules is skewed with a profound effect on B cell maturation.