Soroush Sharbati

Monocytes, a type of leukocytes, are key players in immune responses, transitioning to macrophages at infection sites. Differentiation, which is crucial for macrophage survival and resistance to apoptosis, is a tightly regulated process. Based on our earlier findings implicating the 5'-fragment of tRNA^HisGUG (5'-HisGUG) in macrophage biology, we specifically focused on these fragments to investigate its regulation during monocyte-to-macrophage differentiation and its role in macrophage survival. We performed small RNA sequencing during monocyte-to-macrophage differentiation using THP-1 cells and identified 5'-HisGUG as a prominently regulated tDR. Notably, transfection of 5'-HisGUG in macrophages decreased survival under apoptotic stress. We propose that 5'-HisGUG can also act as an obstructive RNA (oRNA), and its reduction is critical for macrophage survival under stress.

Background: 3D cell culture models have recently garnered increasing attention for replicating organ microarchitecture and eliciting in vivo-like responses, holding significant promise across various biological disciplines. Broadly, 3D cell culture encompasses organoids as well as single- and multicellular spheroids. While the latter have found successful applications in tumor research, there is a notable scarcity of standardized intestinal models for infection biology that mimic the microarchitecture of the intestine. Hence, this study aimed to develop structured multicellular intestinal spheroids (SMIS) specifically tailored for studying molecular basis of infection by intestinal pathogens. Results: We have successfully engineered human SMIS comprising four relevant cell types, featuring a fibroblast core enveloped by an outer monolayer of enterocytes and goblet cells along with monocytic cells. These SMIS effectively emulate the in vivo architecture of the intestinal mucosal surface and manifest differentiated morphological characteristics, including the presence of microvilli, within a mere two days of culture. Through analysis of various differentiation factors, we have illustrated that these spheroids attain heightened levels of differentiation compared to 2D monolayers. Moreover, SMIS serve as an optimized intestinal infection model, surpassing the capabilities of traditional 2D cultures, and exhibit a regulatory pattern of immunological markers similar to in vivo infections after Campylobacter jejuni infection. Notably, our protocol extends beyond human spheroids, demonstrating adaptability to other species such as mice and pigs. Conclusion: Based on the rapid attainment of enhanced differentiation states, coupled with the emergence of functional brush border features, increased cellular complexity, and replication of the intestinal mucosal microarchitecture, which allows for exposure studies via the medium, we are confident that our innovative SMIS model surpasses conventional cell culture methods as a superior model. Moreover, it offers advantages over stem cell-derived organoids due to scalability and standardization capabilities of the protocol. By showcasing differentiated morphological attributes, our model provides an optimal platform for diverse applications. Furthermore, the investigated differences of several immunological factors compared to monotypic monolayers after Campylobacter jejuni infection underline the refinement of our spheroid model, which closely mimics important features of in vivo infections.

Background: Zoonotic microorganisms are increasingly impacting human health worldwide. Due to the development of the global population, humans and animals live in shared and progressively crowded ecosystems, which enhances the risk of zoonoses. Although Campylobacter species are among the most important bacterial zoonotic agents worldwide, the molecular mechanisms of many host and pathogen factors involved in colonisation and infection are poorly understood. Campylobacter jejuni colonises the crypts of the human colon and causes acute inflammatory processes. The mucus and associated proteins play a central host-protective role in this process. The aim of this study was to explore the regulation of specific glycosyltransferase genes relevant to differential mucin-type O-glycosylation that could influence host colonisation and infection by C. jejuni. Results: Since microRNAs are known to be important regulators of the mammalian host cell response to bacterial infections, we focussed on the role of miR-125a-5p in C. jejuni infection. Combining in vitro and in vivo approaches, we show that miR-125a-5p regulates the expression of the sialyltransferase ST3GAL1 in an infection-dependent manner. The protein ST3GAL1 shows markedly increased intestinal levels in infected mice, with enhanced distribution in the mucosal epithelial layer in contrast to naïve mice. Conclusion: From our previous studies and the data presented here, we conclude that miR-125a-5p and the previously reported miR-615-3p are involved in regulating the glycosylation patterns of relevant host cell response proteins during C. jejuni infection. The miRNA-dependent modulation of mucin-type O-glycosylation could be part of the mucosal immune response, but also a pathogen-driven modification that allows colonisation and infection of the mammalian host.

The western honey bee Apis mellifera is globally distributed due to its beekeeping advantages and plays an important role in the global ecology and economy. In recent decades, several studies have raised concerns about bee decline. Discussed are multiple reasons such as increased pathogen pressure, malnutrition or pesticide use. Insecticides are considered to be one of the major factors. In 2013, the use of three neonicotinoids in the field was prohibited in the EU. Flupyradifurone was introduced as a potential successor; it has a comparable mode of action as the banned neonicotinoids. However, there is a limited number of studies on the effects of sublethal concentrations of flupyradifurone on honey bees. Particularly, the larval physiological response by means of protein expression has not yet been studied. Hence, the larval protein expression was investigated via 2D gel electrophoresis after following a standardised protocol to apply sublethal concentrations of the active substance (flupyradifurone 10 mg/kg diet) to larval food. The treated larvae did not show increased mortality or an aberrant development. Proteome comparisons showed clear differences concerning the larval metabolism, immune response and energy supply. Further field studies are needed to validate the in vitro results at a colony level.

Honey bees are important managed pollinators that fulfill important ecological and economic functions. In recent decades, the obligate ectoparasite Varroa destructor severely affected the survival of honey bees, as it weakened them by different means. A common treatment against V. destructor is formic acid fumigation, which has been used for decades by beekeepers across the world. This treatment is known to be effective, but many beekeepers report adverse effects of formic acid on bees, which include damage to the brood, worker bee mortality, and queen loss. Little is known about the molecular mechanisms of formic acid detoxification in honey bees. Recently, we reported upregulation of the bee enzyme, 10-formyl-THFDH, under formic acid fumigation. Here, the active site of this enzyme is characterized by an interdisciplinary approach combining homology modeling and protein mutagenesis. In addition, the limitations of the 3D protein structure prediction program AlphaFold2 are shown in regard to docking studies. This study provides a more thorough understanding of the molecular detoxification mechanisms of formic acid in Apis mellifera.

Background: Campylobacter jejuni (C. jejuni) infections are of increasing importance worldwide. As a typical mucosal pathogen, the interaction of C. jejuni with mucins is a prominent step in the colonisation of mucosal surfaces. Despite recent advances in understanding the interaction between bacterial pathogens and host mucins, the mechanisms of mucin glycosylation during intestinal C. jejuni infection remain largely unclear. This prompted us to identify relevant regulatory networks that are concerted by miRNAs and could play a role in the mucin modification and interaction. Results: We firstly used a human intestinal in vitro model, in which we observed altered transcription of MUC2 and TFF3 upon C. jejuni NCTC 11168 infection. Using a combined approach consisting of in silico analysis together with in vitro expression analysis, we identified the conserved miRNAs miR-125a-5p and miR-615-3p associated with MUC2 and TFF3. Further pathway analyses showed that both miRNAs appear to regulate glycosyltransferases, which are related to the KEGG pathway ‘Mucin type O-glycan biosynthesis’. To validate the proposed interactions, we applied an in vivo approach utilising a well-established secondary abiotic IL-10^−/− mouse model for infection with C. jejuni 81-176. In colonic tissue samples, we confirmed infection-dependent aberrant transcription of MUC2 and TFF3. Moreover, two predicted glycosyltransferases, the sialyltransferases ST3GAL1 and ST3GAL2, exhibited inversely correlated transcriptional levels compared to the expression of the identified miRNAs miR-125a-5p and miR-615-3p, respectively. In this study, we mainly focused on the interaction between miR-615-3p and ST3GAL2 and were able to demonstrate their molecular interaction using luciferase reporter assays and RNAi. Detection of ST3GAL2 in murine colonic tissue by immunofluorescence demonstrated reduced intensity after C. jejuni 81-176 infection and was thus consistent with the observations made above. Conclusions: We report here for the first time the regulation of glycosyltransferases by miRNAs during murine infection with C. jejuni 81-176. Our data suggest that mucin type O-glycan biosynthesis is concerted by the interplay of miRNAs and glycosyltransferases, which could determine the shape of intestinal glycosylated proteins during infection.

Toll-like receptors (TLRs) play a crucial role in the innate immune response. Although endosomal TLR7 recognizes single-stranded RNAs, their endogenous RNA ligands have not been fully explored. Here, we report 5′-tRNA half molecules as abundant activators of TLR7. Mycobacterial infection and accompanying surface TLR activation up-regulate the expression of 5′-tRNA half molecules in human monocyte-derived macrophages (HMDMs). The abundant accumulation of 5′-tRNA halves also occur in HMDM-secreted extracellular vehicles (EVs); the abundance of EV-5′-tRNA^HisGUGhalf molecules is >200-fold higher than that of the most abundant EV-microRNA (miRNA). Sequence identification of the 5′-tRNA halves using cP-RNA-seq revealed abundant and selective packaging of specific 5′-tRNA half species into EVs. The EV-5′-tRNA^HisGUGhalf was experimentally demonstrated to be delivered into endosomes in recipient cells and to activate endosomal TLR7. Up-regulation of the 5′-tRNA half molecules was also observed in the plasma of patients infected withMycobacterium tuberculosis. These results unveil a novel tRNA-engaged pathway in the innate immune response and assign the role of “immune activators” to 5′-tRNA half molecules.

In a recent one-year feeding study, we observed no adverse effects on tissue level in organs of rats fed with the genetically-modified maize MON810. Here, we assessed RNA expression levels of 86 key genes of the apoptosis-, NF-кB-, DNA-damage response (DDR)-, and unfolded-protein response (UPR) pathways by RT-qPCR in the rat liver. Male and female rats were fed either with 33% MON810 (GMO), isogenic- (ISO), or conventional maize (CONV) and RNAs were quantified from eight rats from each of the six feeding groups. Only Birc2 transcript showed a significant (p ≤ 0.05) consistent difference of ≥1.5-fold between the GMO and ISO groups in both sexes. Unsupervised cluster analysis showed a strong separation of male and female rats, but no clustering of the feeding groups. Individual analysis of the pathways did not show any clustering of the male or female feeding groups either, though transcript levels of UPR pathway-associated genes caused some clustering of the male GMO and CONV feeding group samples. These differences were not seen between the GMO and ISO control or within the female cohort. Our data therefore does not support an adverse effect on rat liver RNA expression through the long-term feeding of MON810 compared to isogenic control maize.

Pathogenic mycobacteria are able to persist intracellularly in macrophages, whereas non-pathogenic mycobacteria are effectively combated and eliminated after their phagocytosis. It is known that TGF-β plays an important role in this context. Infection with pathogenic mycobacteria such as Mycobacterium tuberculosis or M. avium leads to production of active TGF-β, which blocks the ability of IFN-γ and TNF-α to inhibit intracellular replication. On the other hand, it is known that the long non-coding RNA (lncRNA) maternally expressed 3 (MEG3) is involved in the regulation of TGF-β. In this study, we show how the infection of THP-1-derived human macrophages with the saprophytic M. smegmatis but not with the facultatively pathogenic M. avium subsp. hominissuis leads to increased MEG3 expression. This is associated with the downregulation of DNA methyltransferases (DNMT) 1 and 3b, which are known to regulate MEG3 expression via promoter hypermethylation. Consequently, we observe a significant downregulation of TGF-β in M. smegmatis-infected macrophages but not in M. avium subsp. hominissuis pointing to lncRNAs as novel mediators of host cell response during mycobacterial infections.

Paratuberculosis is a major disease in cattle that severely affects animal welfare and causes huge economic losses worldwide. Development of alternative diagnostic methods is of urgent need to control the disease. Recent studies suggest that long non-coding RNAs (lncRNAs) play a crucial role in regulating immune function and may confer valuable information about the disease. However, their role has not yet been investigated in cattle with respect to infection towards Paratuberculosis. Therefore, we investigated the alteration in genomic expression profiles of mRNA and lncRNA in bovine macrophages in response to Paratuberculosis infection using RNA-Seq. We identified 397 potentially novel lncRNA candidates in macrophages of which 38 were differentially regulated by the infection. A total of 820 coding genes were also significantly altered by the infection. Co-expression analysis of lncRNAs and their neighbouring coding genes suggest regulatory functions of lncRNAs in pathways related to immune response. For example, this included protein coding genes such asTNIP3,TNFAIP3andNF-κB2 that play a role in NF-κB2 signalling, a pathway associated with immune response. This study advances our understanding of lncRNA roles during Paratuberculosis infection.