Bat ASC2 suppresses inflammasomes and ameliorates inflammatory diseases.

Bats are special in their ability to live long and host many emerging viruses. Our previous studies showed that bats have altered inflammasomes, which are central players in aging and infection. However, the role of inflammasome signaling in combating inflammatory diseases remains poorly understood. Here, we report bat ASC2 as a potent negative regulator of inflammasomes. Bat ASC2 is highly expressed at both the mRNA and protein levels and is highly potent in inhibiting human and mouse inflammasomes. Transgenic expression of bat ASC2 in mice reduced the severity of peritonitis induced by gout crystals and ASC particles. Bat ASC2 also dampened inflammation induced by multiple viruses and reduced mortality of influenza A virus infection. Importantly, it also suppressed SARS-CoV-2-immune-complex-induced inflammasome activation. Four key residues were identified for the gain of function of bat ASC2. Our results demonstrate that bat ASC2 is an important negative regulator of inflammasomes with therapeutic potential in inflammatory diseases.

Establishment of a Captive Cave Nectar Bat (Eonycteris spelaea) Breeding Colony in Singapore.

Bats are known natural reservoirs of several highly pathogenic zoonotic viruses, including Hendra virus, Nipah virus, rabies virus, SARS-like coronaviruses, and suspected ancestral reservoirs of SARS-CoV-2 responsible for the ongoing COVID-19 pandemic. The capacity to survive infections of highly pathogenic agents without severe disease, together with many other unique features, makes bats an ideal animal model for studying the regulation of infection, cancer, and longevity, which is likely to translate into human health outcomes. A key factor that limits bat research is lack of breeding bat colonies. To address this need, a captive bat colony was established in Singapore from 19 wild-caught local cave nectar bats. The bats were screened for specific pathogens before the start of captive breeding. Custom-made cages and an optimized diet inclusive of Wombaroo dietary formula, liquid diet, and supplement of fruits enabled the bats to breed prolifically in our facility. Cages are washed daily and disinfected once every fortnight. Bats are observed daily to detect any sick bat or abnormal behavior. In addition, bats undergo a thorough health check once every 3 to 4 mo to check on their overall wellbeing, perform sampling, and document any potential pregnancy. The current colony houses over 80 bats that are successfully breeding, providing a valuable resource for research in Singapore and overseas.

Extramedullary hematopoiesis: mesenchymal stromal cells from spleen provide an in vitro niche for myelopoiesis.

Murine spleen has been shown to harbour stromal cells that support hematopoiesis with production of myeloid antigen-presenting cells. Similar stromal lines have now been isolated from long-term cultures (LTC) of human spleen. When human progenitor populations from spleen, bone marrow and cord blood were employed as a source of progenitors for co-culture above splenic stromal lines, myelopoiesis was supported. Human splenocytes gave production of predominantly myeloid dendritic-like cells, with minor subsets resembling conventional dendritic cells (cDC) cells, and myeloid or monocyte-derived DC. Human bone marrow progenitors gave rise to myelopoiesis from hematopoietic progenitors, while human cord blood supported limited myelopoiesis from existing myeloid precursors. Transcriptome analysis compared two stromal lines differing in myelopoietic support capacity. Gene profiling revealed both stromal lines to reflect perivascular reticular cells with osteogenic characteristics. However, the 5C6 stroma which failed to support hematopoiesis uniquely expressed several inhibitors of the WNT pathway. Combined data now show that splenic stroma of both human and murine origin provides a mesenchymal stromal cell microenvironment which is WNT pathway-dependent, and which supports in vitro myelopoiesis with production of specific subsets of myeloid and dendritic-like cells.

Culture, expansion, and flow-cytometry-based functional analysis of pteropid bat MR1-restricted unconventional T cells.

Bats harbor viruses of global public health significance. Understanding bat immune systems may provide intervention strategies to prevent zoonotic disease transmission and identify therapeutic targets. This protocol describes how to culture and expand pteropid bat unconventional T cells, restricted by the MHC-I-related protein 1 (MR1), an MHC-I-like protein. Using multicolor flow-cytometry-based techniques, we examine pteropid MR1T cell functionality, including proliferative capacity, cytotoxicity, and cytokine production. This protocol can be adapted to aid immunological research in other bat species. For complete details on the use and execution of this protocol, please refer to Leeansyah et al. (2020b).

MR1-Restricted T Cells with MAIT-like Characteristics Are Functionally Conserved in the Pteropid Bat Pteropus alecto.

Bats are reservoirs for a large number of viruses which have potential to cause major human disease outbreaks, including the current coronavirus disease 2019 (COVID-19) pandemic. Major efforts are underway to understand bat immune response to viruses, whereas much less is known about their immune responses to bacteria. In this study, MR1-restricted T (MR1T) cells were detected through the use of MR1 tetramers in circulation and tissues of Pteropus alecto (Pa) bats. Pa MR1T cells exhibited weak responses to MR1-presented microbial metabolites at resting state. However, following priming with MR1-presented agonist they proliferated, upregulated critical transcription factors and cytolytic proteins, and gained transient expression of Th1/17-related cytokines and antibacterial cytotoxicity. Collectively, these findings show that the Pa bat immune system encompasses an abundant and functionally conserved population of MR1T cells with mucosal-associated invariant T-like characteristics, suggesting that MR1 and MR1T cells also play a significant role in bat immune defense.

Immunophenotyping monocytes, macrophages and granulocytes in the Pteropodid bat Eonycteris spelaea.

Bats are asymptomatic reservoir hosts for several highly pathogenic viruses. Understanding this enigmatic relationship between bats and emerging zoonotic viruses requires tools and approaches which enable the comparative study of bat immune cell populations and their functions. We show that bat genomes have a conservation of immune marker genes which delineate phagocyte populations in humans, while lacking key mouse surface markers such as Ly6C and Ly6G. Cross-reactive antibodies against CD44, CD11b, CD14, MHC II, and CD206 were multiplexed to characterize circulating monocytes, granulocytes, bone-marrow derived macrophages (BMDMs) and lung alveolar macrophages (AMs) in the cave nectar bat Eonycteris spelaea. Transcriptional profiling of bat monocytes and BMDMs identified additional markers - including MARCO, CD68, CD163, CD172α, and CD88 - which can be used to further characterize bat myeloid populations. Bat cells often resembled their human counterparts when comparing immune parameters that are divergent between humans and mice, such as the expression patterns of certain immune cell markers. A genome-wide comparison of immune-related genes also revealed a much closer phylogenetic relationship between bats and humans compared to rodents. Taken together, this study provides a set of tools and a comparative framework which will be important for unravelling viral disease tolerance mechanisms in bats.

A novel myeloid cell in murine spleen defined through gene profiling.

A novel myeloid antigen presenting cell can be generated through in vitro haematopoiesis in long-term splenic stromal cocultures. The in vivo equivalent subset was recently identified as phenotypically and functionally distinct from the spleen subsets of macrophages, conventional (c) dendritic cells (DC), resident monocytes, inflammatory monocytes and eosinophils. This novel subset which is myeloid on the basis of cell surface phenotype, but dendritic-like on the basis of cell surface marker expression and antigen presenting function, has been tentatively labelled "L-DC." Transcriptome analysis has now been employed to determine the lineage relationship of this cell type with known splenic cDC and monocyte subsets. Principal components analysis showed separation of "L-DC" and monocytes from cDC subsets in the second principal component. Hierarchical clustering then indicated a close lineage relationship between this novel subset, resident monocytes and inflammatory monocytes. Resident monocytes were the most closely aligned, with no genes specifically expressed by the novel subset. This subset, however, showed upregulation of genes reflecting both dendritic and myeloid lineages, with strong upregulation of several genes, particularly CD300e. While resident monocytes were found to be dependent on Toll-like receptor signalling for development and were reduced in number in Myd88-/- and Trif-/- mutant mice, both the novel subset and inflammatory monocytes were unaffected. Here, we describe a novel myeloid cell type closely aligned with resident monocytes in terms of lineage but distinct in terms of development and functional capacity.

Studies on B Cells in the Fruit-Eating Black Flying Fox (Pteropus alecto).

The ability of bats to act as reservoir for viruses that are highly pathogenic to humans suggests unique properties and functional characteristics of their immune system. However, the lack of bat specific reagents, in particular antibodies, has limited our knowledge of bat's immunity. Here, we report a panel of cross-reactive antibodies against MHC-II, NK1.1, CD3, CD21, CD27, and immunoglobulin (Ig), that allows flow cytometry analysis of B, T and NK cell populations in two different fruit-eating bat species namely, Pteropus alecto and E. spelaea. Results confirmed predominance of T cells in the spleen and blood of bats, as previously reported by us. However, the percentages of B cells in bone marrow and NK cells in spleen varied greatly between wild caught P. alecto bats and E. spelaea colony bats, which may reflect inherent differences of their immune system or different immune status. Other features of bat B cells were investigated. A significant increase in sIg+ B cell population was observed in the spleen and blood from LPS-injected bats but not from poly I:C-injected bats, supporting T-independent polyclonal B cell activation by LPS. Furthermore, using an in vitro calcium release assay, P. alecto B cells exhibited significant calcium release upon cross-linking of their B cell receptor. Together, this work contributes to improve our knowledge of bat adaptive immunity in particular B cells.

Bat-mouse bone marrow chimera: a novel animal model for dissecting the uniqueness of the bat immune system.

Bats are an important animal model with long lifespans, low incidences of tumorigenesis and an ability to asymptomatically harbour pathogens. Currently, in vivo studies of bats are hampered due to their low reproduction rates. To overcome this, we transplanted bat cells from bone marrow (BM) and spleen into an immunodeficient mouse strain NOD-scid IL-2R-/- (NSG), and have successfully established stable, long-term reconstitution of bat immune cells in mice (bat-mice). Immune functionality of our bat-mouse model was demonstrated through generation of antigen-specific antibody response by bat cells following immunization. Post-engraftment of total bat BM cells and splenocytes, bat immune cells survived, expanded and repopulated the mouse without any observable clinical abnormalities. Utilizing bat's remarkable immunological functions, this novel model has a potential to be transformed into a powerful platform for basic and translational research.

Impact of the c-MybE308G mutation on mouse myelopoiesis and dendritic cell development.

Booreana mice carrying the c-Myb308G point mutation were analyzed to determine changes in early hematopoiesis in the bone marrow and among mature cells in the periphery. This point mutation led to increased numbers of early hematopoietic stem and progenitor cells (HSPCs), with a subsequent reduction in the development of B cells, erythroid cells, and neutrophils, and increased numbers of myeloid cells and granulocytes. Myelopoiesis was further investigated by way of particular subsets affected. A specific question addressed whether booreana mice contained increased numbers of dendritic-like cells (L-DC subset) recently identified in the spleen, since L-DCs arise in vitro by direct differentiation from HSPCs co-cultured over splenic stroma. The non-lethal c-Myb mutation in booreana mice was associated with significantly lower representation of splenic CD8- conventional dendritic cells (cDCs), inflammatory monocytes, and neutrophils compared to wild-type mice. This result confirmed the bone marrow origin of progenitors for these subsets since c-Myb is essential for their development. Production of L-DCs and resident monocytes was not affected by the c-MybE308G mutation. These subsets may derive from different progenitors than those in bone marrow, and are potentially established in the spleen during embryogenesis. An alternative explanation may be needed for why there was no change in CD8+ cDCs in booreana spleen since these cells are known to derive from common dendritic progenitors in bone marrow.

Antigen presenting capacity of murine splenic myeloid cells.

BACKGROUND: The spleen is an important site for hematopoiesis. It supports development of myeloid cells from bone marrow-derived precursors entering from blood. Myeloid subsets in spleen are not well characterised although dendritic cell (DC) subsets are clearly defined in terms of phenotype, development and functional role. Recently a novel dendritic-like cell type in spleen named 'L-DC' was distinguished from other known dendritic and myeloid cells by its distinct phenotype and developmental origin. That study also redefined splenic eosinophils as well as resident and inflammatory monocytes in spleen. RESULTS: L-DC are shown to be distinct from known splenic macrophages and monocyte subsets. Using a new flow cytometric procedure, it has been possible to identify and isolate L-DC in order to assess their functional competence and ability to activate T cells both in vivo and in vitro. L-DC are readily accessible to antigen given intravenously through receptor-mediated endocytosis. They are also capable of CD8+ T cell activation through antigen cross presentation, with subsequent induction of cytotoxic effector T cells. L-DC are MHCII- cells and unable to activate CD4+ T cells, a property which clearly distinguishes them from conventional DC. The myeloid subsets of resident monocytes, inflammatory monocytes, neutrophils and eosinophils, were found to have varying capacities to take up antigen, but were uniformly unable to activate either CD4+ T cells or CD8+ T cells. CONCLUSION: The results presented here demonstrate that L-DC in spleen are distinct from other myeloid cells in that they can process antigen for CD8+ T cell activation and induction of cytotoxic effector function, while both L-DC and myeloid subsets remain unable to activate CD4+ T cells. The L-DC subset in spleen is therefore distinct as an antigen presenting cell.

Antigen Presenting Properties of a Myeloid Dendritic-Like Cell in Murine Spleen.

This paper distinguishes a rare subset of myeloid dendritic-like cells found in mouse spleen from conventional (c) dendritic cells (DC) in terms of phenotype, function and gene expression. These cells are tentatively named "L-DC" since they resemble dendritic-like cells produced in longterm cultures of spleen. L-DC can be distinguished on the basis of their unique phenotype as CD11bhiCD11cloMHCII-CD43+Ly6C-Ly6G-Siglec-F- cells. They demonstrate similar ability as cDC to uptake and retain complex antigens like mannan via mannose receptors, but much lower ability to endocytose and retain soluble antigen. While L-DC differ from cDC by their inability to activate CD4+ T cells, they are capable of antigen cross-presentation for activation of CD8+ T cells, although less effectively so than the cDC subsets. In terms of gene expression, CD8- cDC and CD8+ cDC are quite distinct from L-DC. CD8+ cDC are distinguishable from the other two subsets by expression of CD24a, Clec9a, Xcr1 and Tlr11, while CD8- cDC are distinguished by expression of Ccnd1 and H-2Eb2. L-DC are distinct from the two cDC subsets through upregulated expression of Clec4a3, Emr4, Itgam, Csf1r and CD300ld. The L-DC gene profile is quite distinct from that of cDC, confirming a myeloid cell type with distinct antigen presenting properties.

Redefining Myeloid Cell Subsets in Murine Spleen.

Spleen is known to contain multiple dendritic and myeloid cell subsets, distinguishable on the basis of phenotype, function and anatomical location. As a result of recent intensive flow cytometric analyses, splenic dendritic cell (DC) subsets are now better characterized than other myeloid subsets. In order to identify and fully characterize a novel splenic subset termed "L-DC" in relation to other myeloid cells, it was necessary to investigate myeloid subsets in more detail. In terms of cell surface phenotype, L-DC were initially characterized as a CD11b(hi)CD11c(lo)MHCII(-)Ly6C(-)Ly6G(-) subset in murine spleen. Their expression of CD43, lack of MHCII, and a low level of CD11c was shown to best differentiate L-DC by phenotype from conventional DC subsets. A complete analysis of all subsets in spleen led to the classification of CD11b(hi)CD11c(lo)MHCII(-)Ly6C(lo)Ly6G(-) cells as monocytes expressing CX3CR1, CD43 and CD115. Siglec-F expression was used to identify a specific eosinophil population, distinguishable from both Ly6C(lo) and Ly6C(hi) monocytes, and other DC subsets. L-DC were characterized as a clear subset of CD11b(hi)CD11c(lo)MHCII(-)Ly6C(-)Ly6G(-) cells, which are CD43(+), Siglec-F(-) and CD115(-). Changes in the prevalence of L-DC compared to other subsets in spleens of mutant mice confirmed the phenotypic distinction between L-DC, cDC and monocyte subsets. L-DC development in vivo was shown to occur independently of the BATF3 transcription factor that regulates cDC development, and also independently of the FLT3L and GM-CSF growth factors which drive cDC and monocyte development, so distinguishing L-DC from these commonly defined cell types.

Spleen stroma maintains progenitors and supports long-term hematopoiesis.

Hematopoietic stem/progenitor cells (HSPC) differentiate in the context of stromal niches producing cells of multiple lineages. Limited success has been achieved in the past with induction of hematopoiesis in vitro. Previously, spleen long-term stromal cultures (LTC) were shown to continuously support restricted hematopoiesis for production of novel dendritic-like cells (LTC-DC). An in vivo equivalent dendritic cell type was then described which is specific for spleen. The in vivo counterpart cell was termed 'L-DC' and represents a dendritic-like CD11c(lo)CD11b(hi)CD8α-MHC-II- cell which differs phenotypically and functionally from monocytes/macrophages and conventional and plasmacytoid DC. Splenic stroma is now shown to maintain HSPC and to support their restricted in vitro differentiation to give this 'L-DC' subset. In order to characterise progenitors of this distinct cell type, LTC were analysed for cell subsets produced, and these subsets sorted and assessed for hematopoietic potential in subsequent co-cultures over STX3 stroma. Progenitors were defined as a lineage (Lin)(-)ckit(lo) subset reflecting HSPC. Furthermore, when Lin(-)ckit(hi)Sca1(+)Flt3(-) HSPC were sorted from bone marrow, they colonised splenic stroma with long-term production of L-DC. The maintenance of HSPC by splenic stroma was confirmed when non-adherent cells collected from LTC showed oligopotent reconstitution of the hematopoietic compartment of lethally irradiated mice. All data support a model whereby spleen houses a niche for HSPC in the resting state, with production of progenitors, and their differentiation to give tissue-specific antigen presenting cells.

Spleen as a site for hematopoiesis of a distinct antigen presenting cell type.

While spleen and other secondary tissue sites contribute to hematopoiesis, the nature of cells produced and the environment under which this happens are not fully defined. Evidence is reviewed here for hematopoiesis occurring in the spleen microenvironment leading to the production of tissue-specific antigen presenting cells. The novel dendritic-like cell identified in spleen is phenotypically and functionally distinct from other described antigen presenting cells. In order to identify these cells as distinct, it has been necessary to show that their lineage origin and progenitors differ from that of other known dendritic and myeloid cell types. The spleen therefore represents a distinct microenvironment for hematopoiesis of a novel myeloid cell arising from self-renewing hematopoietic stem cells (HSC) or progenitors endogenous to spleen.

Identification of a novel antigen cross-presenting cell type in spleen.

Antigen-presenting cells (APC), like dendritic cells (DC), are essential for T-cell activation, leading to immunity or tolerance. Multiple DC subsets each play a unique role in the immune response. Here, a novel splenic dendritic-like APC has been characterized in mice that has immune function and cell surface phenotype distinct from other, described DC subsets. These were identified as a cell type continuously produced in spleen long-term cultures (LTC) and have an in vivo equivalent cell type in mice, namely 'L-DC'. This study characterizes LTC-DC in terms of marker phenotype and function, and compares them with L-DC and other known splenic DC and myeloid subsets. L-DC display a myeloid dendritic-like phenotype equivalent to LTC-DC as CD11c(lo) CD11b(hi) MHC-II(-) CD8α(-) cells, distinct by high accessibility and endocytic capacity for blood-borne antigen. Both LTC-DC and L-DC have strong antigen cross-presentation ability leading to strong activation of CD8(+) T cells, particularly after exposure to lipopolysaccharide. However, they have weak ability to stimulate CD4(+) T cells in antigen-specific responses. Evidence is presented here for a novel DC type produced by in vitro haematopoiesis which has distinct antigen-presenting potential and reflects a DC subset present also in vivo in spleen.