CTLA-4 Modulates the Differentiation of Inducible Foxp3+ Treg Cells but IL-10 Mediates Their Function in Experimental Autoimmune Encephalomyelitis

In vitro induced Foxp3⁺ T regulatory (iTreg) cells form a novel and promising target for therapeutic tolerance induction. However, the potential of these cells as a target for the treatment of various immune diseases, as well as the factors involved in their development and function, remain debated. Here, we demonstrate in a myelin basic protein (MBP)-specific murine model of CNS autoimmune disease that adoptive transfer of antigen-specific iTreg cells ameliorates disease progression. Moreover, we show that the co-stimulatory molecule CTLA-4 mediates in vitro differentiation of iTreg cells. Finally, we demonstrate that the secreted, immunosuppressive cytokine IL-10 controls the ability of antigen-specific iTreg cells to suppress autoimmune disease. Overall, we conclude that antigen-specific iTreg cells, which depend on various immune regulatory molecules for their differentiation and function, represent a major target for effective immunotherapy of autoimmune disease.     

Effects of double ligation of Stensen's duct on the rabbit parotid gland

Salivary gland duct ligation is an alternative to gland excision for treating sialorrhea or reducing salivary gland size prior to tumor excision. Duct ligation also is used as an approach to study salivary gland aging, regeneration, radiotherapy, sialolithiasis and sialadenitis. Reports conflict about the contribution of each salivary cell population to gland size reduction after ductal ligation. Certain cell populations, especially acini, reportedly undergo atrophy, apoptosis and proliferation during reduction of gland size. Acini also have been reported to de-differentiate into ducts. These contradictory results have been attributed to different animal or salivary gland models, or to methods of ligation. We report here a bilateral double ligature technique for rabbit parotid glands with histologic observations at 1, 7, 14, 30, 60 days after ligation. A large battery of special stains and immunohistochemical procedures was employed to define the cell populations. Four stages with overlapping features were observed that led to progressive shutdown of gland activities: 1) marked atrophy of the acinar cells occurred by 14 days, 2) response to and removal of the secretory material trapped in the acinar and ductal lumens mainly between 30 and 60 days, 3) reduction in the number of parenchymal (mostly acinar) cells by apoptosis that occurred mainly between 14–30 days, and 4) maintenance of steady-state at 60 days with a low rate of fluid, protein, and glycoprotein secretion, which greatly decreased the number of leukocytes engaged in the removal of the luminal contents. The main post- ligation characteristics were dilation of ductal and acinar lumens, massive transient infiltration of mostly heterophils (rabbit polymorphonuclear leukocytes), acinar atrophy, and apoptosis of both acinar and ductal cells. Proliferation was uncommon except in the larger ducts. By 30 days, the distribution of myoepithelial cells had spread from exclusively investing the intercalated ducts pre-ligation to surrounding a majority of the residual duct-like structures, many of which clearly were atrophic acini. Thus, both atrophy and apoptosis made major contributions to the post-ligation reduction in gland size. Structures also occurred with both ductal and acinar markers that suggested acini differentiating into ducts. Overall, the reaction to duct ligation proceeded at a considerably slower pace in the rabbit parotid glands than has been reported for the salivary glands of the rat.     

Propagation of viral genomes by replicating ammonia-oxidising archaea during soil nitrification

Ammonia-oxidising archaea (AOA) are a ubiquitous component of microbial communities and dominate the first stage of nitrification in some soils. While we are beginning to understand soil virus dynamics, we have no knowledge of the composition or activity of those infecting nitrifiers or their potential to influence processes. This study aimed to characterise viruses having infected autotrophic AOA in two nitrifying soils of contrasting pH by following transfer of assimilated CO₂-derived ¹³C from host to virus via DNA stable-isotope probing and metagenomic analysis. Incorporation of ¹³C into low GC mol% AOA and virus genomes increased DNA buoyant density in CsCl gradients but resulted in co-migration with dominant non-enriched high GC mol% genomes, reducing sequencing depth and contig assembly. We therefore developed a hybrid approach where AOA and virus genomes were assembled from low buoyant density DNA with subsequent mapping of ¹³C isotopically enriched high buoyant density DNA reads to identify activity of AOA. Metagenome-assembled genomes were different between the two soils and represented a broad diversity of active populations. Sixty-four AOA-infecting viral operational taxonomic units (vOTUs) were identified with no clear relatedness to previously characterised prokaryote viruses. These vOTUs were also distinct between soils, with 42% enriched in ¹³C derived from hosts. The majority were predicted as capable of lysogeny and auxiliary metabolic genes included an AOA-specific multicopper oxidase suggesting infection may augment copper uptake essential for central metabolic functioning. These findings indicate virus infection of AOA may be a frequent process during nitrification with potential to influence host physiology and activity.Subject terms: Microbial ecology, Stable isotope analysis

Microbially mediated oxidation of ammonia to nitrate during nitrification is a central component of the global nitrogen (N) cycle. It is also responsible for major losses of applied fertiliser N in soil, generating atmospheric pollution via direct and indirect production of nitrous oxide (N₂O) as well as nitrate (NO₃^-) pollution of groundwater [1]. Autotrophic ammonia-oxidising archaea (AOA) of the class Nitrososphaeria are a ubiquitous component of soil microbial communities and often dominate ammonia oxidation and nitrification-associated N₂O emissions when ammonia is supplied at low rates via organic matter mineralisation [2], slow-release fertilisers [3] or in acidic soils [4]. Integrated temperate viruses (proviruses) and other virus-associated protein-encoding genes are found in most AOA genomes suggesting frequent interaction (see Supplementary Text). While viruses infecting marine AOA have been characterised through metagenomic approaches [5] and cultivation [6], those infecting soil AOA or other nitrifier groups are currently uncharacterised.Virus infection can influence biogeochemical cycling by augmenting host activity or causing cell mortality and subsequent release of nutrients [7]. Recent advances have demonstrated that soil virus communities are dynamic in a wide range of soils [e.g. 8, 9] and augmenting virus loads modulate C and N fluxes [10, 11]. Nevertheless, identifying active interactions with specific populations or functional groups in soil remains challenging due to structural complexity and the vast diversity of hosts and viruses. Recent use of stable-isotope approaches has investigated whole community host-virus dynamics [12, 13] or interactions between individual host-virus populations specific to a functional process and substrate [14]. The aim of this study was to utilise the latter approach with ¹³CO₂-based DNA-SIP to focus on nitrification-associated interactions and to test the hypothesis that viruses are a dynamic component of soil AOA activity.     

DNA processing by the Kaposi's sarcoma-associated herpesvirus alkaline exonuclease SOX contributes to viral gene expression and infectious virion production

Alkaline exonucleases (AE) are present in several large DNA viruses including bacteriophage λ and herpesviruses, where they play roles in viral DNA processing during genome replication. Given the genetic conservation of AEs across viruses infecting different kingdoms of life, these enzymes likely assume central roles in the lifecycles of viruses where they have yet to be well characterized. Here, we applied a structure-guided functional analysis of the bifunctional AE in the oncogenic human gammaherpesvirus Kaposi''s sarcoma-associated herpesvirus (KSHV), called SOX. In addition to identifying a preferred DNA substrate preference for SOX, we define key residues important for DNA binding and DNA processing, and how SOX activity on DNA partially overlaps with its functionally separable cleavage of mRNA. By engineering these SOX mutants into KSHV, we reveal roles for its DNase activity in viral gene expression and infectious virion production. Our results provide mechanistic insight into gammaherpesviral AE activity as well as areas of functional conservation between this mammalian virus AE and its distant relative in phage λ.     

Genome of Enterobacteriophage Lula/phi80 and Insights into Its Ability To Spread in the Laboratory Environment

The novel temperate bacteriophage Lula, contaminating laboratory Escherichia coli strains, turned out to be the well-known lambdoid phage phi80. Our previous studies revealed that two characteristics of Lula/phi80 facilitate its spread in the laboratory environment: cryptic lysogen productivity and stealthy infectivity. To understand the genetics/genomics behind these traits, we sequenced and annotated the Lula/phi80 genome, encountering an E. coli-toxic gene revealed as a gap in the sequencing contig and analyzing a few genes in more detail. Lula/phi80''s genome layout copies that of lambda, yet homology with other lambdoid phages is mostly limited to the capsid genes. Lula/phi80''s DNA is resistant to cutting with several restriction enzymes, suggesting DNA modification, but deletion of the phage''s damL gene, coding for DNA adenine methylase, did not make DNA cuttable. The damL mutation of Lula/phi80 also did not change the phage titer in lysogen cultures, whereas the host dam mutation did increase it almost 100-fold. Since the high phage titer in cultures of Lula/phi80 lysogens is apparently in response to endogenous DNA damage, we deleted the only Lula/phi80 SOS-controlled gene, dinL. We found that dinL mutant lysogens release fewer phage in response to endogenous DNA damage but are unchanged in their response to external DNA damage. The toxic gene of Lula/phi80, gamL, encodes an inhibitor of the host ATP-dependent exonucleases, RecBCD and SbcCD. Its own antidote, agt, apparently encoding a modifier protein, was found nearby. Interestingly, Lula/phi80 lysogens are recD and sbcCD phenocopies, so GamL and Agt are part of lysogenic conversion.