The Drosophila lymph gland, the larval hematopoietic organ comprised of prohemocytes and mature hemocytes, has been a valuable model for understanding mechanisms underlying hematopoiesis and immunity. Three types of mature hemocytes have been characterized in the lymph gland: plasmatocytes, lamellocytes, and crystal cells, which are analogous to vertebrate myeloid cells, yet molecular underpinnings of the lymph gland hemocytes have been less investigated. Here, we use single-cell RNA sequencing to comprehensively analyze heterogeneity of developing hemocytes in the lymph gland, and discover previously undescribed hemocyte types including adipohemocytes, stem-like prohemocytes, and intermediate prohemocytes. Additionally, we identify the developmental trajectory of hemocytes during normal development as well as the emergence of the lamellocyte lineage following active cellular immunity caused by wasp infestation. Finally, we establish similarities and differences between embryonically derived- and larval lymph gland hemocytes. Altogether, our study provides detailed insights into the hemocyte development and cellular immune responses at single-cell resolution.

Drosophila hemocytes, like those of mammals, are given rise from two distinctive phases during both the embryonic and larval hematopoiesis. Embryonically derived hemocytes, mostly composed of macrophage-like plasmatocytes, are largely identified by genetic markers. However, the cellular diversity and distinct functions of possible subpopulations within plasmatocytes have not been explored in Drosophila larvae. Here, we show that larval plasmatocytes exhibit differential expressions of Hemolectin (Hml) and Peroxidasin (Pxn) during development. Moreover, removal of plasmatocytes by overexpressing pro-apoptotic genes, hid and reaper in Hml-positive plasmatocytes, feeding high sucrose diet, or wasp infestation results in increased circulating hemocytes that are Hml-negative. Interestingly these Hml-negative plasmatocytes retain Pxn expression, and animals expressing Hml-negative and Pxn-positive subtype largely attenuate growth and abrogate metabolism. Furthermore, elevated levels of a cytokine, unpaired 3, are detected when Hml-positive hemocytes are ablated, which in turn activates JAK/STAT activity in several tissues including the fat body. Finally, we observed that insulin signaling is inhibited in this background, which can be recovered by concurrent loss of upd3. Overall, this study highlights heterogeneity in Drosophila plasmatocytes and a functional plasticity of each subtype, which reaffirms extension of their role beyond immunity into metabolic regulation for cooperatively maintaining internal homeostatic balance.

SUMMARYDrosophila lymph gland, the larval hematopoietic organ comprised of prohemocytes and hemocytes, has been a valuable model for understanding mechanisms underlying hematopoiesis and immunity. Three types of mature hemocytes have been characterized in the lymph gland: plasmatocytes, lamellocytes, and crystal cells, which are analogous to vertebrate myeloid cells. Here, we used single-cell RNA sequencing to comprehensively analyze heterogeneity of developing hemocytes in the lymph gland, and discovered novel hemocyte types, stem-like prohemocytes, and intermediate prohemocytes. Additionally, we identified the emergence of the lamellocyte lineage following active cellular immunity caused by wasp infestation. We unraveled similarities and differences between embryonically derived- and larval lymph gland hemocytes. Finally, the comparison of Drosophila lymph gland hemocytes and human immune cells highlights similarities between prohemocytes and hematopoietic stem cell, and between mature hemocytes and myeloid cells across species. Altogether, our study provides detailed insights on the development and evolution of hematopoiesis at single-cell resolution.

Drosophila melanogaster lymph gland, the primary site of hematopoiesis, contains myeloid-like progenitor cells that differentiate into functional hemocytes in the circulation of pupae and adults. Fly hemocytes are dynamic and plastic, and they play diverse roles in the innate immune response and wound healing. Various hematopoietic regulators in the lymph gland ensure the developmental and functional balance between progenitors and mature blood cells. In addition, systemic factors, such as nutrient availability and sensory inputs, integrate environmental variabilities to synchronize the blood development in the lymph gland with larval growth, physiology, and immunity. This review examines the intrinsic and extrinsic factors determining the progenitor states during hemocyte development in the lymph gland and provides new insights for further studies that may extend the frontier of our collective knowledge on hematopoiesis and innate immunity.

Background To facilitate the drive towards Universal Health Coverage (UHC) several countries in the West African subregion have over the last two decades adopted the system of National Health Insurance (NHI) to finance their health services. However, most of these countries continue to face challenges safeguarding the insured population against catastrophic health expenditure (CHE) and impoverishment due to health spending. The aim of this study is to describe the extent of financial risk protection among households enrolled under NHI schemes in West Africa and summarize potential learnings. Methods We conducted a systematic review of observational studies in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Studies published in English between 2005 and 2022 were searched for using keywords, synonyms and MeSH terms related to NHI, financial risk protection and UHC in all West African countries on the following electronic databases: PubMed/Medline, Web of Science and CINAHL via EBSCOhost and Embase via Ovid and Google Scholar. The quality of included studies was assessed using the Joanna Briggs Institute (JBI) critical appraisal checklist. Two independent reviewers assessed the studies for inclusion, extracted data and conducted quality assessment. We present the findings of the narrative synthesis consisting of thematic synthesis for qualitative data and a Synthesis Without Meta-analysis (SWiM) for quantitative data. The study protocol was published in PROSPERO under the ID CRD42022338574 on 28th June 2022. Results Of the 1,279 articles initially identified, nine were eligible for inclusion. These were cross-sectional studies (n=8) and retrospective cohort study (n=1) published between 2011 and 2021 in Ghana (n=8) and Nigeria (n=1). Two-thirds of the included studies reported that enrollment into the NHI showed a positive (protective) effect on CHE at different thresholds and one study showed a protective effect of NHI on impoverishment due to health spending. However, almost all of the included studies (n=8) reported that a proportion of insured households still encountered CHE with one-third of them reporting more than 50% of insured households incurring CHE. Key determinants of CHE and impoverishment due to health spending reported consisted of income, employment and educational status of household members as well as household size, household health profile, gender of household head and distance of household from health facility. Discussion Households insured under NHI schemes in some West African countries (Ghana and Nigeria) are better protected against CHE and impoverishment due to health spending compared to uninsured households as evidenced in other studies. However, insured households continue to incur CHE and impoverishment due to health expenditure resulting from gaps identified in the current design of NHI schemes in these West African countries. Conclusion To protect insured households from the financial burden due to health spending and advance the drive towards UHC in West Africa, governments should consider investing more into research on NHI, implementing nationwide compulsory NHI programmes and establishing a multinational West African collaboration to co-design a sustainable contextspecific NHI system based on solidarity, equity and fairness in financial contribution.