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Mitochondrial genome variation affects humoral and cell-mediated innate immune responses and infection outcomes

ABSTRACT: The role of mitochondria in both adaptive and innate immune responses is increasingly recognized, but the role of natural mitochondrial DNA (mtDNA) variation as an immunomodulatory factor has received less attention. One reason for this is the difficulty of separating the effect of mtDNA from that of the nuclear genome. By utilizing the fruit fly Drosophila melanogaster, a powerful model system, we created cytoplasmic hybrids, aka. cybrid lines, where unique mtDNAs (mitotypes) were introgressed into a controlled isogenic nuclear background. We harnessed a panel of cybrid lines to study the effect of mtDNA variation on humoral and cell-mediated innate immune responses. Mitotypes exhibited heterogeneity in infection outcomes upon bacterial, viral and parasitoid infections. One mitotype of note, mtKSA2 appeared to be more immunocompetent when compared to other mitotypes. We performed transcriptomic profiling of uninfected flies and flies infected with bacteria or a virus and compared them to a different mitotype in the same nuclear background to find the mechanistic basis of the immunocompetence. We found that mtKSA2 caused an upregulation of TCA and OXPHOS related genes and a downregulation of a multiple genes encoding for antimicrobial peptides in uninfected flies. Upon infection, mtKSA2 flies produced unique transcriptomes that were separated by infection type and duration. When we examined immune cells (hemocytes) in mtKSA2 larvae, we noted an increase in hemocyte numbers. These hemocytes were activated in the absence of infection, increased their production of ROS, and showed evidence of increased encapsulation efficiency upon parasitoid wasp infection. Overall, our results show that mtDNA variation acts as an immunomodulatory factor in both humoral and cell-mediated innate immunity and that specific mitotypes provide enhanced protection against infections.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE260986 | GEO | 2024/03/09

REPOSITORIES: GEO

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Project description:Although host-parasitoid interactions are becoming well characterized at the organismal and cellular levels, much remains to be understood of the molecular bases for the host immune response and the parasitoidsâ ability to defeat this immune response. Leptopilina boulardi and L. heterotoma, two closely related, highly infectious natural parasitoids of Drosophila melanogaster, appear to use very different infection strategies at the cellular level. Here, we further characterize cellular level differences in the infection characteristics of these two wasp species using newly derived, virulent inbred strains, and then use whole genome microarrays to compare the transcriptional response of Drosophila to each. While flies attacked by the melanogaster group specialist Leptopilina boulardi (strain Lb17) up-regulate numerous genes encoding proteolytic enzymes, components of the Toll and JAK/STAT pathways, and the melanization cascade as part of a combined cellular and humoral innate immune response, flies attacked by the generalist L. heterotoma (strain Lh14) do not appear to initiate an immune transcriptional response at the time points post-infection we assayed, perhaps due to the rapid venom-mediated lysis of host hemocytes (blood cells). Thus, the specialist parasitoid appears to invoke a full-blown immune response in the host, but suppresses and/or evades downstream components of this response. Given that activation of the host immune response likely depletes the energetic resources of the host, the specialistâs infection strategy seems relatively disadvantageous. However, we uncover the mechanism for one potentially important fitness tradeoff of the generalistâs highly immune suppressive infection strategy. Experiment Overall Design: The parasitoid wasps L. boulardi and L. heterotoma were allowed to attack late second instar D. melanogaster larvae (72 hrs old at 22ËC) in the following manner. Nine petri dishes containing 60 fly larvae were each exposed to six experienced L. boulardi (strain Lb17) female wasps for 2 hrs, another nine plates were exposed to five L. heterotoma (strain Lh14) females, and nine control plates were left uninfected. For each of three time points post-infection (2-5 hrs, 9-12 hrs, 21-24 hrs), 40 larvae from three replicate plates were removed and frozen at -80ËC for RNA extraction and microarray analysis (3 treatments x 3 time points x 3 replicates = 27 samples total).

Project description:The mitochondrial and nuclear genomes contribute to mitochondrial function, and when mitochondrial function is compromised, mitochondrial retrograde signaling alters nuclear gene expression. We performed gene expression profiling of engineered cells that had mitochondria containing a disease-associated mutation that causes mitochondrial dysfunction. By generating networks of transcription factors that targeted these genes, the authors revealed putative mitochondrial retrograde signaling pathways. One such pathway involved retinoic acid receptor alpha (RXRA), the mRNA for which was reduced in the mutant cells. Network analysis and experiments in cells suggested that mitochondrial dysfunction caused by the mutation initiated a positive feedback loop that aggravated mitochondrial dysfunction: Reduced RXRA abundance further compromised expression of genes encoding products involved in mitochondrial function and translation. This gene-transcription factor mapping-network approach may reveal targets for therapeutic intervention of diseases associated with mitochondrial dysfunction. To investigate retrograde signaling pathways induced by the mitochondrial dysfunction caused by the mt3243 mutation, we generated the three types of cybrid cells using a mitochondria-mediated transformation method. Cells lacking mtDNA (rho0) were fused with mtDNAs with the mt3243 mutation isolated from platelets of a diabetic patient with sensorineural hearing loss. We then isolated three types of cybrid cells: W cells had wild-type mtDNA (3243A homoplasmy), H cells had both the mutant and wild-type mtDNA (3243A/G heteroplasmy) with 70% of the mtDNA containing 3243G, and M cells with only mutant mtDNA (3243G homoplasmy). Gene expression profiles of cybrid cells were generated using Illumina HumanHT-12-v3-BeadChip (Illumina, San Diego, CA), which includes 49,896 probes corresponding to 25,202 annotated genes. According to the Illumina protocols, three biological triplicates of each type of cybrid cells were analyzed. Total RNA (500ng) was isolated from cybrid cells using RNeasy Mini Kit (Qiagen, GmbH, Germany). RNA integrity number (RIN) was in the range of RIN = 9.2 and 10 when measured with an Agilent 2100 Bioanalyzer. RNA was reversely transcribed and amplified using IlluminaTotalPrep RNA amplification kit (Ambion, Austin, TX). In vitro transcription was then carried out to prepare cRNA. The cRNAs were hybridized to the array and then labeled with Cy3-streptavidin (Amersham Bioscience, Little Chalfont, UK). The fluorescent signal on the array was measured with a BeadStation 500 System (Illumina, San Diego, CA).

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Mitochondrial genome variation affects humoral and cell-mediated innate immune responses and infection outcomes

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