Macrophage transactivation for chemokine production identified as a negative regulator of granulomatous inflammation using agent-based modeling

Cellular activation in trans by interferons, cytokines and chemokines is a commonly recognized mechanism to amplify immune effector function and limit pathogen spread. However, an optimal host response also requires that collateral damage associated with inflammation is limited. This may be particularly so in the case of granulomatous inflammation, where an excessive number and / or excessively florid granulomas can have significant pathological consequences. Here, we have combined transcriptomics, agent-based modeling and in vivo experimental approaches to study constraints on hepatic granuloma formation in a murine model of experimental leishmaniasis. We demonstrate that chemokine production by non-infected Kupffer cells in the Leishmania donovani-infected liver promotes competition with infected KCs for available iNKT cells, ultimately inhibiting the extent of granulomatous inflammation. We propose trans-activation for chemokine production as a novel broadly applicable mechanism that may operate early in infection to limit excessive focal inflammation

Plasmodium Strain Determines Dendritic Cell Function Essential for Survival from Malaria

The severity of malaria can range from asymptomatic to lethal infections involving severe anaemia and cerebral disease. However, the molecular and cellular factors responsible for these differences in disease severity are poorly understood. Identifying the factors that mediate virulence will contribute to developing antiparasitic immune responses. Since immunity is initiated by dendritic cells (DCs), we compared their phenotype and function following infection with either a nonlethal or lethal strain of the rodent parasite, Plasmodium yoelii, to identify their contribution to disease severity. DCs from nonlethal infections were fully functional and capable of secreting cytokines and stimulating T cells. In contrast, DCs from lethal infections were not functional. We then transferred DCs from mice with nonlethal infections to mice given lethal infections and showed that these DCs mediated control of parasitemia and survival. IL-12 was necessary for survival. To our knowledge, our studies have shown for the first time that during a malaria infection, DC function is essential for survival. More importantly, the functions of these DCs are determined by the strain of parasite. Our studies may explain, in part, why natural malaria infections may have different outcomes

Raster adaptive optics for video rate aberration correction and large FOV multiphoton imaging

Removal of complex aberrations at millisecond time scales over millimeters in distance in multiphoton laser scanning microscopy limits the total spatiotemporal imaging throughput for deep tissue imaging. Using a single low resolution deformable mirror and time multiplexing (TM) adaptive optics, we demonstrate video rate aberration correction (5 ms update rate for a single wavefront mask) for a complex heterogeneous distribution of refractive index differences through a depth of up to 1.1 mm and an extended imaging FOV of up to 0.8 mm, with up to 167% recovery of fluorescence intensity 335 µm from the center of the FOV. The proposed approach, termed raster adaptive optics (RAO), integrates image-based aberration retrieval and video rate removal of arbitrarily defined regions of dominant, spatially varied wavefronts. The extended FOV was achieved by demonstrating rapid recovery of up to 50 distinct wavefront masks at 500 ms update rates that increased imaging throughput by 2.3-fold. Because RAO only requires a single deformable mirror with image-based aberration retrieval, it can be directly implemented on a standard laser scanning multiphoton microscope.Australian Research Council (CE140100011, DE160100843, DP190100039)

IFNAR1-Signalling Obstructs ICOS-mediated Humoral Immunity during Non-lethal Blood-Stage Plasmodium Infection

Funding: This work was funded by a Career Development Fellowship (1028634) and a project grant (GRNT1028641) awarded to AHa by the Australian National Health & Medical Research Council (NHMRC). IS was supported by The University of Queensland Centennial and IPRS Scholarships. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Bone marrow-derived and resident liver macrophages display unique transcriptomic signatures but similar biological functions

Abstract: Background and aims: Kupffer cells (KCs), the resident tissue macrophages of the liver, play a crucial role in the clearance of pathogens and other particulate materials that reach the systemic circulation. Recent studies have identified KCs as a yolk sac-derived resident macrophage population that is replenished independently of monocytes in the steady state. Although it is now established that following local tissue injury, bone-marrow derived monocytes may infiltrate the tissue and differentiate into macrophages, the extent to which newly differentiated macrophages functionally resemble the KCs they have replaced has not been extensively studied. Methods and results: Here we show using intravital microscopy, morphometric analysis and gene expression profiling that bone marrow derived “KCs” accumulating as a result of genotoxic injury resemble, but are not identical to their yolk-sac (YS) counterparts. An ion homeostasis gene signature, including genes associated with scavenger receptor function and extracellular matrix deposition, allows discrimination between these two KC populations. Reflecting the differential expression of scavenger receptors, YS-derived KCs were more effective at accumulating Ac-LDL, whereas surprisingly they were poorer than BM-derived KCs when assessed for uptake of a range of bacterial pathogens. The two KC populations were almost indistinguishable in regard to i) response to LPS challenge, ii) phagocytosis of effete RBCs and iii) their ability to contain infection and direct granuloma formation against Leishmania donovani, a KC-tropic intracellular parasite. Conclusions: BM-derived KCs differentiate locally to resemble YS-derived KC in most but not all respects, with implications for models of infectious diseases, liver injury and bone marrow transplantation. In addition, the gene signature we describe adds to the tools available for distinguishing KC subpopulations based on their ontology

B Cell: T Cell Interactions Occur within Hepatic Granulomas during Experimental Visceral Leishmaniasis

Hepatic resistance to Leishmania donovani infection in mice is associated with the development of granulomas, in which a variety of lymphoid and non-lymphoid populations accumulate. Although previous studies have identified B cells in hepatic granulomas and functional studies in B cell-deficient mice have suggested a role for B cells in the control of experimental visceral leishmaniasis, little is known about the behaviour of B cells in the granuloma microenvironment. Here, we first compared the hepatic B cell population in infected mice, where ≈60% of B cells are located within granulomas, with that of naïve mice. In infected mice, there was a small increase in mIgMlomIgD+ mature B2 cells, but no enrichment of B cells with regulatory phenotype or function compared to the naïve hepatic B cell population, as assessed by CD1d and CD5 expression and by IL-10 production. Using 2-photon microscopy to quantify the entire intra-granuloma B cell population, in conjunction with the adoptive transfer of polyclonal and HEL-specific BCR-transgenic B cells isolated from L. donovani-infected mice, we demonstrated that B cells accumulate in granulomas over time in an antigen-independent manner. Intra-vital dynamic imaging was used to demonstrate that within the polyclonal B cell population obtained from L. donovani-infected mice, the frequency of B cells that made multiple long contacts with endogenous T cells was greater than that observed using HEL-specific B cells obtained from the same inflammatory environment. These data indicate, therefore, that a subset of this polyclonal B cell population is capable of making cognate interactions with T cells within this unique environment, and provide the first insights into the dynamics of B cells within an inflammatory site

In vivo imaging of systemic transport and elimination of xenobiotics and endogenous molecules in mice

We describe a two-photon microscopy-based method to evaluate the in vivo systemic transport of compounds. This method comprises imaging of the intact liver, kidney and intestine, the main organs responsible for uptake and elimination of xenobiotics and endogenous molecules. The image quality of the acquired movies was sufficient to distinguish subcellular structures like organelles and vesicles. Quantification of the movement of fluorescent dextran and fluorescent cholic acid derivatives in different organs and their sub-compartments over time revealed significant dynamic differences. Calculated half-lives were similar in the capillaries of all investigated organs but differed in the specific sub-compartments, such as parenchymal cells and bile canaliculi of the liver, glomeruli, proximal and distal tubules of the kidney and lymph vessels (lacteals) of the small intestine. Moreover, tools to image immune cells, which can influence transport processes in inflamed tissues, are described. This powerful approach provides new possibilities for the analysis of compound transport in multiple organs and can support physiologically based pharmacokinetic modeling, in order to obtain more precise predictions at the whole body scale

The Neurotrophic Receptor Ntrk2 Directs Lymphoid Tissue Neovascularization during Leishmania donovani Infection

The neurotrophic tyrosine kinase receptor type 2 (Ntrk2, also known as TrkB) and its ligands brain derived neurotrophic factor (Bdnf), neurotrophin-4 (NT-4/5), and neurotrophin-3 (NT-3) are known primarily for their multiple effects on neuronal differentiation and survival. Here, we provide evidence that Ntrk2 plays a role in the pathologic remodeling of the spleen that accompanies chronic infection. We show that in Leishmania donovani-infected mice, Ntrk2 is aberrantly expressed on splenic endothelial cells and that new maturing blood vessels within the white pulp are intimately associated with F4/80hiCD11bloCD11c+ macrophages that express Bdnf and NT-4/5 and have pro-angiogenic potential in vitro. Furthermore, administration of the small molecule Ntrk2 antagonist ANA-12 to infected mice significantly inhibited white pulp neovascularization but had no effect on red pulp vascular remodeling. We believe this to be the first evidence of the Ntrk2/neurotrophin pathway driving pathogen-induced vascular remodeling in lymphoid tissue. These studies highlight the therapeutic potential of modulating this pathway to inhibit pathological angiogenesis

Innate Killing of Leishmania donovani by Macrophages of the Splenic Marginal Zone Requires IRF-7

Highly phagocytic macrophages line the marginal zone (MZ) of the spleen and the lymph node subcapsular sinus. Although these macrophages have been attributed with a variety of functions, including the uptake and clearance of blood and lymph-borne pathogens, little is known about the effector mechanisms they employ after pathogen uptake. Here, we have combined gene expression profiling and RNAi using a stromal macrophage cell line with in situ analysis of the leishmanicidal activity of marginal zone macrophages (MZM) and marginal metallophilic macrophages (MMM) in wild type and gene targeted mice. Our data demonstrate a critical role for interferon regulatory factor-7 (IRF-7) in regulating the killing of intracellular Leishmania donovani by these specialised splenic macrophage sub-populations. This study, therefore, identifies a new role for IRF-7 as a regulator of innate microbicidal activity against this, and perhaps other, non-viral intracellular pathogens. This study also highlights the importance of selecting appropriate macrophage populations when studying pathogen interactions with this functionally diverse lineage of cells

The role of the spleen in Malaria : Cellular changes that affect the development of immunity

Malaria, caused by the apicomplexan parasite Plasmodium, is a major cause of morbidity and mortality throughout the world. This study has focused on the role of the spleen in the control of the blood stage of infection. Three aspects have been examined specifically: the effect of infection on the architecture of the spleen, the role of the spleen in parasite clearance and the formation of B cell memory. Firstly, the effect of infection on the splenic microarchitecture was examined. An essential component of the splenic architecture is the marginal zone (MZ), an area of the spleen that separates the reticuloendothelial red pulp of the spleen from the lymphoid white pulp compartment. Two unique populations of macrophages are found in the marginal zone: marginal zone macrophages (MZM) and marginal metallophilic macrophages (MMM). In the current study, parasitised red blood cells (pRBC) as well as normal RBC located to the MZ thirty minutes after intravenous injection and formed close associations with both MMM and MZM. Eight days after infection, at the time of peak parasitemia, a complete loss of both MMM and MZM was observed. Assays to detect cell death revealed that the loss of both MMM and MZM appeared to occur as a result of apoptosis. The apoptosis was not induced by up regulation of the inflammatory cytokines tumour necrosis factor or interferon-γ and could not be blocked by over expression of the apoptosis inhibitor Bcl2. Significantly, MMM were retained in the absence of CD8+ T cells implicating CD8+ T cells in the loss of MMM. Finally, infection of CD95-/- mice demonstrated that CD95/CD95-ligand (Fas/Fas-ligand) interactions were responsible for some of the CD8+ T cell-mediated loss of MMM. These data provide evidence for a novel interaction between MMM and CD8+ T cellsfollowing infection with Plasmodium. Secondly, the role of the spleen in the control of parasitemia and disease was monitored with an emphasis on determining the role of splenic macrophage populations (MMM, MZM and red pulp macrophages [RPM]) in parasite clearance. A clodronate liposome-mediated macrophage depletion technique was used, and caused a complete loss of all three macrophage sub-populations, as well as 50% of splenic dendritic cells, within 24 hours of administration. Each of the macrophage populations, as well as splenic DC, demonstrated different repopulation kinetics following their depletion from the spleen and these kinetics were utilised to examine each cell population in isolation. RPM depleted mice had significantly higher peak parasitemias than the controls. This peak returned to the level observed in undepleted control animals only after the repopulation of RPM was complete, suggesting that RPM play a role in the control of peak parasitemia following infection. Neither MMM nor MZM played a role in the control of parasitemia. The role of non-splenic macrophages and splenic dendritic cells also was investigated and shown to be insignificant in the absence of splenic macrophages. Finally, the role of RPM in mice immune to infection was investigated and their role shown to be dispensable, with immune mice clearing parasitemia efficiently in the absence of RPM. RPM therefore are important for the innate control of infection with P. chabaudi but are dispensible once adaptive immunity is established. Finally, the role of the spleen in the development of parasite-specific B cell memory was examined. Initial studies demonstrated that germinal centre (GC) development was compromised following infection with P. chabaudi, with an involution of B cell follicles noted early in infection. Adoptive transfer of memory B cells from immunised to naïve mice demonstrated that some protection was conferred on recipient mice by parasite-specific memory B cells. But, the memory B cells could not protect the host from developing parasitemia and did not produce significant amounts of parasite-specific immunoglobulin within seven days of challenge infection. Memory B cells could not be detected ten weeks after infection, indicating that the development, or survival, of parasite-specific memory B cells was compromised. The development of bystander memory B cells was not affected by infection. Finally, long-lived plasma cells were shown to develop in response to infection, although re-exposure of the cells to parasites in the form of recrudescent parasitemia resulted in their loss. This study therefore has identified a defect in the development of long-term, B cell-mediated, protection against infection with P. chabaudi. Each of these factors has significant implications for the understanding of how the spleen contributes to the control of infection with Plasmodium and potential applications for the further development of malaria vaccines and treatment regimens