Plasmacytoid dendritic cells appear inactive during sub-microscopic Plasmodium falciparum blood-stage infection, yet retain their ability to respond to TLR stimulation

Plasmacytoid dendritic cells (pDC) are activators of innate and adaptive immune responses that express HLA-DR, toll-like receptor (TLR) 7, TLR9 and produce type I interferons. The role of human pDC in malaria remains poorly characterised. pDC activation and cytokine production were assessed in 59 malaria-naive volunteers during experimental infection with 150 or 1,800 P. falciparum-parasitized red blood cells. Using RNA sequencing, longitudinal changes in pDC gene expression were examined in five adults before and at peak-infection. pDC responsiveness to TLR7 and TLR9 stimulation was assessed in-vitro. Circulating pDC remained transcriptionally stable with gene expression altered for 8 genes (FDR < 0.07). There was no upregulation of co-stimulatory molecules CD86, CD80, CD40, and reduced surface expression of HLA-DR and CD123 (IL-3R-α). pDC loss from the circulation was associated with active caspase-3, suggesting pDC apoptosis during primary infection. pDC remained responsive to TLR stimulation, producing IFN-α and upregulating HLA-DR, CD86, CD123 at peak-infection. In clinical malaria, pDC retained HLA-DR but reduced CD123 expression compared to convalescence. These data demonstrate pDC retain function during a first blood-stage P. falciparum exposure despite sub-microscopic parasitaemia downregulating HLA-DR. The lack of evident pDC activation in both early infection and malaria suggests little response of circulating pDC to infection

Epigenetic Profiling and Molecular Characterisation of Non-melanoma Skin Cancer

PhDNon-melanoma skin (NMSC) cancer is the most common human malignancy. Cutaneous squamous cell carcinoma (cSCC) and its precursor, actinic keratosis (AK) affect tens of thousands of people each year in the UK. Merkel cell carcinoma is a rare, yet aggressive type of NMSC recently linked with Merkel Cell Polyomavirus (MCPyV). In spite of the clinical burden of NMSC, key molecular regulatory patterns remain largely unknown. The aims of this thesis were to investigate genome-wide genetic, epigenetic and transcriptional changes in AK and cSCC, and assess the prevalence of MCPyV and its effect on methylation in NMSC. Copy-number analysis revealed that AK harbours significantly more genomic aberrations compared to skin, the majority of which occurs on chromosomes 8 and 9. Transcriptional profiling has found 292 and 308 genes as differentially expressed in AK compared to non-sunexposed and sun-exposed skin, respectively, and gene-set enrichment analysis (GSEA) revealed dysregulation of PPAR pathway in this lesion. Expression profiling of cSCC and AK has revealed 346 differentially expressed genes, and GSEA detected dysregulation in several canonical pathways including TGF-β and MAPK pathway. Aberrant methylation in cSCC cell lines occurs in the promoters of many developmental genes. A total of 1085 hyper- and 833 hypomethylated genes were detected in cSCCs, and GSEA revealed dysregulation of critical signalling pathways (WNT, MAPK signalling pathways). Methylation analysis of AK revealed a total of 4194 differentially methylated genes, and implicated FOXF2, PITX2, RUNX1 and SMAD3 transcription factors in this lesions. MiRNA profiling of cSCC and normal skin revealed significant dysregulation of 38 miRNAs including several of viral origin. MCPyV was shown to be common in NMSC, yet MCPyV nor human papillomavirus does not affect cSCC methylation. Taken together, this work provides novel insight into molecular regulation of cSCC oncogenesis, and identifies potential epigenetic targets for functional evaluation in this malignancy.British Skin Foundation and the Barts and the London Charity research grant

Profoundly reduced CD1c+myeloid dendritic cell HLA-DR and CD86 expression and increased tumor necrosis factor production in experimental human blood-stage malaria infection

Dendritic cells (DCs) are sentinels of the immune system that uniquely prime naive cells and initiate adaptive immune responses. CD1c (BDCA-1) myeloid DCs (CD1c+ mDCs) highly express HLA-DR, have a broad Toll-like receptor (TLR) repertoire, and secrete immune modulatory cytokines. To better understand immune responses to malaria, CD1c+ mDC maturation and cytokine production were examined in healthy volunteers before and after experimental intravenous Plasmodium falciparum infection with 150- or 1,800-parasite-infected red blood cells (pRBCs). After either dose, CD1c+ mDCs significantly reduced HLA-DR expression in prepatent infections. Circulating CD1c+ mDCs did not upregulate HLA-DR after pRBC or TLR ligand stimulation and exhibited reduced CD86 expression. At peak parasitemia, CD1c+ mDCs produced significantly more tumor necrosis factor (TNF), whereas interleukin-12 (IL-12) production was unchanged. Interestingly, only the 1,800-pRBC dose caused a reduction in the circulating CD1c+ mDC count with evidence of apoptosis. The 1,800-pRBC dose produced no change in T cell IFN-γ or IL-2 production at peak parasitemia or at 3 weeks posttreatment. Overall, CD1c+ mDCs are compromised by P. falciparum exposure, with impaired HLA-DR and CD86 expression, and have an increased capacity for TNF but not IL-12 production. A first prepatent P. falciparum infection is sufficient to modulate CD1c+ mDC responsiveness, likely contributing to hampered effector T cell cytokine responses and assisting parasite immune evasion