Fetal origin confers radioresistance on liver macrophages via p21cip1/WAF1.

BACKGROUND & AIMS: Cells of hematopoietic origin, including macrophages, are generally radiation sensitive, but a subset of Kupffer cells (KCs) is relatively radioresistant. Here, we focused on the identity of the radioresistant KCs in unmanipulated mice and the mechanism of radioresistance. METHODS: We employed Emr1- and inducible CX3Cr1-based fate-mapping strategies combined with the RiboTag reporter to identify the total KCs and the embryo-derived KCs, respectively. The KC compartment was reconstituted with adult bone-marrow-derived KCs (bm-KCs) using clodronate depletion. Mice were lethally irradiated and transplanted with donor bone marrow, and the radioresistance of bone-marrow- or embryo-derived KCs was studied. Gene expression was analyzed using in situ mRNA isolation via RiboTag reporter mice, and the translatomes were compared among subsets. RESULTS: Here, we identified the radioresistant KCs as the long-lived subset that is derived from CX3CR1-expressing progenitor cells in fetal life, while adult bm-KCs do not resist irradiation. While both subsets upregulated the Cdkn1a gene, encoding p21-cip1/WAF1 protein, radioresistant embryo-derived KCs showed a greater increase in response to irradiation. In the absence of this molecule, the radioresistance of KCs was compromised. Replacement KCs, derived from adult hematopoietic stem cells, differed from radioresistant KCs in their expression of genes related to immunity and phagocytosis. CONCLUSIONS: Here, we show that, in the murine liver, a subset of KCs of embryonic origin resists lethal irradiation through Cdkn1a upregulation and is maintained for a long period, while bm-KCs do not survive lethal irradiation. LAY SUMMARY: Kupffer cells (KCs) are the tissue-resident macrophages of the liver. KCs can be originated from fetal precursors and from monocytes during the fetal stage and post-birth, respectively. Most immune cells in mice are sensitive to lethal-irradiation-induced death, while a subset of KCs resists radiation-induced death. These radioresistant KCs continue to live in the irradiated mice. We discovered that this relatively radioresistant KC subset are the fetal-derived KCs, and they achieve this through cell-cycle arrest. Understanding the radiobiology of KCs will provide valuable insights into the mechanisms that elicit radiation-induced liver disease.

A Plasmodium α/ß-hydrolase modulates the development of invasive stages.

The bud emergence (BEM)46 proteins are evolutionarily conserved members of the α/ß-hydrolase superfamily, which includes enzymes with diverse functions and a wide range of substrates. Here, we identified a Plasmodium BEM46-like protein (PBLP) and characterized it throughout the life cycle of the rodent malaria parasite Plasmodium yoelii. The Plasmodium BEM46-like protein is shown to be closely associated with the parasite plasma membrane of asexual erythrocytic stage schizonts and exo-erythrocytic schizonts; however, PBLP localizes to unique intracellular structures in sporozoites. Generation and analysis of P. yoelii knockout (Δpblp) parasite lines showed that PBLP has an important role in erythrocytic stage merozoite development with Δpblp parasites forming fewer merozoites during schizogony, which results in decreased parasitemia when compared with wild-type (WT) parasites. Δpblp parasites showed no defects in gametogenesis or transmission to mosquitoes; however, because they formed fewer oocysts there was a reduction in the number of developed sporozoites in infected mosquitoes when compared with WT. Although Δpblp sporozoites showed no apparent defect in mosquito salivary gland infection, they showed decreased infectivity in hepatocytes in vitro. Similarly, mice infected with Δpblp sporozoites exhibited a delay in the onset of blood-stage patency, which is likely caused by reduced sporozoite infectivity and a discernible delay in exo-erythrocytic merozoite formation. These data are consistent with the model that PBLP has an important role in parasite invasive-stage morphogenesis throughout the parasite life cycle.

Host-based Prophylaxis Successfully Targets Liver Stage Malaria Parasites.

Eliminating malaria parasites during the asymptomatic but obligate liver stages (LSs) of infection would stop disease and subsequent transmission. Unfortunately, only a single licensed drug that targets all LSs, Primaquine, is available. Targeting host proteins might significantly expand the repertoire of prophylactic drugs against malaria. Here, we demonstrate that both Bcl-2 inhibitors and P53 agonists dramatically reduce LS burden in a mouse malaria model in vitro and in vivo by altering the activity of key hepatocyte factors on which the parasite relies. Bcl-2 inhibitors act primarily by inducing apoptosis in infected hepatocytes, whereas P53 agonists eliminate parasites in an apoptosis-independent fashion. In combination, Bcl-2 inhibitors and P53 agonists act synergistically to delay, and in some cases completely prevent, the onset of blood stage disease. Both families of drugs are highly effective at doses that do not cause substantial hepatocyte cell death in vitro or liver damage in vivo. P53 agonists and Bcl-2 inhibitors were also effective when administered to humanized mice infected with Plasmodium falciparum. Our data demonstrate that host-based prophylaxis could be developed into an effective intervention strategy that eliminates LS parasites before the onset of clinical disease and thus opens a new avenue to prevent malaria.

Susceptibility to Plasmodium yoelii preerythrocytic infection in BALB/c substrains is determined at the point of hepatocyte invasion.

After transmission by Anopheles mosquitoes, Plasmodium sporozoites travel to the liver, infect hepatocytes, and rapidly develop as intrahepatocytic liver stages (LS). Rodent models of malaria exhibit large differences in the magnitude of liver infection, both between parasite species and between strains of mice. This has been mainly attributed to differences in innate immune responses and parasite infectivity. Here, we report that BALB/cByJ mice are more susceptible to Plasmodium yoelii preerythrocytic infection than BALB/cJ mice. This difference occurs at the level of early hepatocyte infection, but expression levels of reported host factors that are involved in infection do not correlate with susceptibility. Interestingly, BALB/cByJ hepatocytes are more frequently polyploid; thus, their susceptibility converges on the previously observed preference of sporozoites to infect polyploid hepatocytes. Gene expression analysis demonstrates hepatocyte-specific differences in mRNA abundance for numerous genes between BALB/cByJ and BALB/cJ mice, some of which encode hepatocyte surface molecules. These data suggest that a yet-unknown receptor for sporozoite infection, present at elevated levels on BALB/cByJ hepatocytes and also polyploid hepatocytes, might facilitate Plasmodium liver infection.

A next-generation genetically attenuated Plasmodium falciparum parasite created by triple gene deletion.

Immunization with live-attenuated Plasmodium sporozoites completely protects against malaria infection. Genetic engineering offers a versatile platform to create live-attenuated sporozoite vaccine candidates. We previously generated a genetically attenuated parasite (GAP) by deleting the P52 and P36 genes in the NF54 wild-type (WT) strain of Plasmodium falciparum (Pf p52(-)/p36(-) GAP). Preclinical assessment of p52(-)/p36(-) GAP in a humanized mouse model indicated an early and severe liver stage growth defect. However, human exposure to >200 Pf p52(-)/p36(-) GAP-infected mosquito bites in a safety trial resulted in peripheral parasitemia in one of six volunteers, revealing that this GAP was incompletely attenuated. We have now created a triple gene deleted GAP by additionally removing the SAP1 gene (Pf p52(-)/p36(-)/sap1(-) GAP) and employed flippase (FLP)/flippase recognition target (FRT) recombination for drug selectable marker cassette removal. This next-generation GAP was indistinguishable from WT parasites in blood stage and mosquito stage development. Using an improved humanized mouse model transplanted with human hepatocytes and human red blood cells, we show that despite a high-dose sporozoite challenge, Pf p52(-)/p36(-)/sap1(-) GAP did not transition to blood stage infection and appeared to be completely attenuated. Thus, clinical testing of Pf p52(-)/p36(-)/sap1(-) GAP assessing safety, immunogenicity, and efficacy against sporozoite challenge is warranted.

SSP3 is a novel Plasmodium yoelii sporozoite surface protein with a role in gliding motility.

Plasmodium sporozoites develop within oocysts in the mosquito midgut wall and then migrate to the salivary glands. After transmission, they embark on a complex journey to the mammalian liver, where they infect hepatocytes. Proteins on the sporozoite surface likely mediate multiple steps of this journey, yet only a few sporozoite surface proteins have been described. Here, we characterize a novel, conserved sporozoite surface protein (SSP3) in the rodent malaria parasite Plasmodium yoelii. SSP3 is a putative type I transmembrane protein unique to Plasmodium. By using epitope tagging and SSP3-specific antibodies in conjunction with immunofluorescence microscopy, we showed that SSP3 is expressed in mosquito midgut oocyst sporozoites, exhibiting an intracellular localization. In sporozoites derived from the mosquito salivary glands, however, SSP3 localized predominantly to the sporozoite surface as determined by immunoelectron microscopy. However, the ectodomain of SSP3 appeared to be inaccessible to antibodies in nonpermeabilized salivary gland sporozoites. Antibody-induced shedding of the major surface protein circumsporozoite protein (CSP) exposed the SSP3 ectodomain to antibodies in some sporozoites. Targeted deletion of SSP3 adversely affected in vitro sporozoite gliding motility, which, surprisingly, impacted neither their cell traversal capacity, host cell invasion in vitro, nor infectivity in vivo. Together, these data reveal a previously unappreciated complexity of the Plasmodium sporozoite surface proteome and the roles of surface proteins in distinct biological activities of sporozoites.

Model for in vivo assessment of humoral protection against malaria sporozoite challenge by passive transfer of monoclonal antibodies and immune serum.

Evidence from clinical trials of malaria vaccine candidates suggests that both cell-mediated and humoral immunity to pre-erythrocytic parasite stages can provide protection against infection. Novel pre-erythrocytic antibody (Ab) targets could be key to improving vaccine formulations, which are currently based on targeting antigens such as the circumsporozoite protein (CSP). However, methods to assess the effects of sporozoite-specific Abs on pre-erythrocytic infection in vivo remain underdeveloped. Here, we combined passive transfer of monoclonal Abs (MAbs) or immune serum with a luciferase-expressing Plasmodium yoelii sporozoite challenge to assess Ab-mediated inhibition of liver infection in mice. Passive transfer of a P. yoelii CSP MAb showed inhibition of liver infection when mice were challenged with sporozoites either intravenously or by infectious mosquito bite. However, inhibition was most potent for the mosquito bite challenge, leading to a more significant reduction of liver-stage burden and even a lack of progression to blood-stage parasitemia. This suggests that Abs provide effective protection against a natural infection. Inhibition of liver infection was also achieved by passive transfer of immune serum from whole-parasite-immunized mice. Furthermore, we demonstrated that passive transfer of a MAb against P. falciparum CSP inhibited liver-stage infection in a humanized mouse/P. falciparum challenge model. Together, these models constitute unique and sensitive in vivo methods to assess serum-transferable protection against Plasmodium sporozoite challenge.

Put the Vanc Down, Flip It and Reverse It: Comparison of Vancomycin and Daptomycin Health Care Utilization and Cost in Outpatient Parenteral Antimicrobial Therapy.

Vancomycin and daptomycin are frequently used in outpatient parenteral antimicrobial therapy (OPAT). We analyze health care utilization and cost to the health care system for vancomycin vs daptomycin in the outpatient setting and find that vancomycin results in significantly higher health care utilization and similar cost per course compared with daptomycin in OPAT.

Dalba Got Back? Use of Dalbavancin for the Treatment of Vertebral Osteomyelitis.

Data evaluating dalbavancin use for vertebral osteomyelitis remain limited. In our retrospective cohort, 29 of 34 (85.3%) patients completed their dalbavancin course. Adverse reactions occurred for 6 (17.6%) and infection recurrence in 3 (8.8%) within 90 days. Dalbavancin appears to be safe and well-tolerated for vertebral osteomyelitis.

The hidden cost of dalbavancin: OPAT RN time required in coordination for persons who use drugs.

Background: Serious infections in persons who use drugs (PWUD) are rising. Dalbavancin, due to its extended half-life, offers an alternative treatment for patients in whom standard of care antibiotics are not feasible or practical, allowing for reduced hospital days and the avoidance of central line placement or the use of complex oral regimens. Objectives: We aim to describe the time and effort required for coordination of dalbavancin courses by outpatient registered nurses (RNs) and other outpatient parenteral antimicrobial therapy (OPAT) staff. Design and methods: We conducted a retrospective review of adult patients with documented substance use who received at least one dose of dalbavancin and quantified the number of interventions required by our OPAT RNs and other OPAT staff for coordination of dalbavancin courses. Additionally, detailed data on time spent per intervention were prospectively collected for a 1-month period. Results: A total of 52 patients with 53 dalbavancin courses were included. Most substance use was intravenous. Infectious diagnoses included bone and joint infections (61%) and endocarditis (7%), in addition to skin and soft tissue infections (19%). Infections were most commonly caused by Staphylococcus aureus (62%). RN intervention was required in the coordination of 60% of all courses and in 77% of courses in which at least one outpatient dose was needed. Adverse reactions occurred in one patient (2%) and 90-day readmissions due to infectious complications occurred in two patients (4%). Detailed time analysis was performed for seven consecutive patients, with a total of 179 min spent by OPAT RNs on coordination. Conclusions: The ease of dalbavancin administration does not eliminate the need for extensive RN coordination for successful administration of doses in the outpatient setting for PWUD. This need should be accounted for in program staffing to help increase successful dalbavancin course completion.

A genome-wide CRISPR-Cas9 screen identifies CENPJ as a host regulator of altered microtubule organization during Plasmodium liver infection.

Prior to initiating symptomatic malaria, a single Plasmodium sporozoite infects a hepatocyte and develops into thousands of merozoites, in part by scavenging host resources, likely delivered by vesicles. Here, we demonstrate that host microtubules (MTs) dynamically reorganize around the developing liver stage (LS) parasite to facilitate vesicular transport to the parasite. Using a genome-wide CRISPR-Cas9 screen, we identified host regulators of cytoskeleton organization, vesicle trafficking, and ER/Golgi stress that regulate LS development. Foci of γ-tubulin localized to the parasite periphery; depletion of centromere protein J (CENPJ), a novel regulator identified in the screen, exacerbated this re-localization and increased infection. We demonstrate that the Golgi acts as a non-centrosomal MT organizing center (ncMTOC) by positioning γ-tubulin and stimulating MT nucleation at parasite periphery. Together, these data support a model where the Plasmodium LS recruits host Golgi to form MT-mediated conduits along which host organelles are recruited to PVM and support parasite development.

Liver stage malaria infection is controlled by host regulators of lipid peroxidation.

The facets of host control during Plasmodium liver infection remain largely unknown. We find that the SLC7a11-GPX4 pathway, which has been associated with the production of reactive oxygen species, lipid peroxidation, and a form of cell death called ferroptosis, plays a critical role in control of Plasmodium liver stage infection. Specifically, blocking GPX4 or SLC7a11 dramatically reduces Plasmodium liver stage parasite infection. In contrast, blocking negative regulators of this pathway, NOX1 and TFR1, leads to an increase in liver stage infection. We have shown previously that increased levels of P53 reduces Plasmodium LS burden in an apoptosis-independent manner. Here, we demonstrate that increased P53 is unable to control parasite burden during NOX1 or TFR1 knockdown, or in the presence of ROS scavenging or when lipid peroxidation is blocked. Additionally, SLC7a11 inhibitors Erastin and Sorafenib reduce infection. Thus, blocking the host SLC7a11-GPX4 pathway serves to selectively elevate lipid peroxides in infected cells, which localize within the parasite and lead to the elimination of liver stage parasites.

Flow Cytometry-Based Assessment of Antibody Function Against Malaria Pre-erythrocytic Infection.

The development of new interventional strategies against pre-erythrocytic malaria is hampered by the lack of standardized approaches to assess inhibition of sporozoite infection of hepatocytes. The following methodology, based on flow cytometry, can be used to quantitatively assess P. falciparum sporozoite infection in vitro in medium throughput. In addition to assessing the efficacy of antibodies, this assay has a wide variety of applications for investigating basic science questions about the malaria liver stage. This approach is easily applied in a variety of laboratory settings, assesses the functionality of antibody responses against malaria sporozoites, and can be adapted for the limited quantities of sample which are typically available from clinical investigations.

Malaria parasites target the hepatocyte receptor EphA2 for successful host infection.

The invasion of a suitable host hepatocyte by mosquito-transmitted Plasmodium sporozoites is an essential early step in successful malaria parasite infection. Yet precisely how sporozoites target their host cell and facilitate productive infection remains largely unknown. We found that the hepatocyte EphA2 receptor was critical for establishing a permissive intracellular replication compartment, the parasitophorous vacuole. Sporozoites productively infected hepatocytes with high EphA2 expression, and the deletion of EphA2 protected mice from liver infection. Lack of host EphA2 phenocopied the lack of the sporozoite proteins P52 and P36. Our data suggest that P36 engages EphA2, which is likely to be a key step in establishing the permissive replication compartment.

Suppression of host p53 is critical for Plasmodium liver-stage infection.

Plasmodium parasites infect the liver and replicate inside hepatocytes before they invade erythrocytes and trigger clinical malaria. Analysis of host signaling pathways affected by liver-stage infection could provide critical insights into host-pathogen interactions and reveal targets for intervention. Using protein lysate microarrays, we found that Plasmodium yoelii rodent malaria parasites perturb hepatocyte regulatory pathways involved in cell survival, proliferation, and autophagy. Notably, the prodeath protein p53 was substantially decreased in infected hepatocytes, suggesting that it could be targeted by the parasite to foster survival. Indeed, mice that express increased levels of p53 showed reduced liver-stage parasite burden, whereas p53 knockout mice suffered increased liver-stage burden. Furthermore, boosting p53 levels with the use of the small molecule Nutlin-3 dramatically reduced liver-stage burden in vitro and in vivo. We conclude that perturbation of the hepatocyte p53 pathway critically impacts parasite survival. Thus, host pathways might constitute potential targets for host-based antimalarial prophylaxis.