Murine alveolar macrophages rapidly accumulate intranasally administered SARS-CoV-2 Spike protein leading to neutrophil recruitment and damage.

The trimeric SARS-CoV-2 Spike protein mediates viral attachment facilitating cell entry. Most COVID-19 vaccines direct mammalian cells to express the Spike protein or deliver it directly via inoculation to engender a protective immune response. The trafficking and cellular tropism of the Spike protein in vivo and its impact on immune cells remains incompletely elucidated. In this study, we inoculated mice intranasally, intravenously, and subcutaneously with fluorescently labeled recombinant SARS-CoV-2 Spike protein. Using flow cytometry and imaging techniques, we analyzed its localization, immune cell tropism, and acute functional impact. Intranasal administration led to rapid lung alveolar macrophage uptake, pulmonary vascular leakage, and neutrophil recruitment and damage. When injected near the inguinal lymph node medullary, but not subcapsular macrophages, captured the protein, while scrotal injection recruited and fragmented neutrophils. Widespread endothelial and liver Kupffer cell uptake followed intravenous administration. Human peripheral blood cells B cells, neutrophils, monocytes, and myeloid dendritic cells all efficiently bound Spike protein. Exposure to the Spike protein enhanced neutrophil NETosis and augmented human macrophage TNF-α (tumor necrosis factor-α) and IL-6 production. Human and murine immune cells employed C-type lectin receptors and Siglecs to help capture the Spike protein. This study highlights the potential toxicity of the SARS-CoV-2 Spike protein for mammalian cells and illustrates the central role for alveolar macrophage in pathogenic protein uptake.

The V2 domain of HIV gp120 mimics an interaction between CD4 and integrin ⍺4ß7.

The CD4 receptor, by stabilizing TCR-MHC II interactions, plays a central role in adaptive immunity. It also serves as the HIV docking receptor. The HIV gp120 envelope protein binds directly to CD4. This interaction is a prerequisite for viral entry. gp120 also binds to ⍺4ß7, an integrin that is expressed on a subset of memory CD4+ T cells. HIV tropisms for CD4+ T cells and gut tissues are central features of HIV pathogenesis. We report that CD4 binds directly to ⍺4ß7 in a dynamic way, consistent with a cis regulatory interaction. The molecular details of this interaction are related to the way in which gp120 interacts with both receptors. Like MAdCAM-1 and VCAM-1, two recognized ligands of ⍺4ß7, the binding interface on CD4 includes 2 sites (1° and accessory), distributed across its two N-terminal IgSF domains (D1 and D2). The 1° site includes a sequence in the G ß-strand of CD4 D2, KIDIV, that binds directly to ⍺4ß7. This pentapeptide sequence occurs infrequently in eukaryotic proteins. However, a closely related and conserved sequence, KLDIV, appears in the V2 domain of gp120. KLDIV mediates gp120-⍺4ß7 binding. The accessory ⍺4ß7 binding site on CD4 includes Phe43. The Phe43 aromatic ring protrudes outward from one edge of a loop connecting the C'C" strands of CD4 D1. Phe43 is a principal contact for HIV gp120. It interacts with conserved residues in the recessed CD4 binding pocket. Substitution of Phe43 abrogates CD4 binding to both gp120 and ⍺4ß7. As such, the interactions of gp120 with both CD4 and ⍺4ß7 reflect elements of their interactions with each other. These findings indicate that gp120 specificities for CD4 and ⍺4ß7 are interrelated and suggest that selective pressures which produced a CD4 tropic virus that replicates in gut tissues are linked to a dynamic interaction between these two receptors.

Murine Alveolar Macrophages Rapidly Accumulate Intranasally Administered SARS-CoV-2 Spike Protein leading to Neutrophil Recruitment and Damage.

The trimeric SARS-CoV-2 Spike protein mediates viral attachment facilitating cell entry. Most COVID-19 vaccines direct mammalian cells to express the Spike protein or deliver it directly via inoculation to engender a protective immune response. The trafficking and cellular tropism of the Spike protein in vivo and its impact on immune cells remains incompletely elucidated. In this study we inoculated mice intranasally, intravenously, and subcutaneously with fluorescently labeled recombinant SARS-CoV-2 Spike protein. Using flow cytometry and imaging techniques we analyzed its localization, immune cell tropism, and acute functional impact. Intranasal administration led to rapid lung alveolar macrophage uptake, pulmonary vascular leakage, and neutrophil recruitment and damage. When injected near the inguinal lymph node medullary, but not subcapsular macrophages, captured the protein, while scrotal injection recruited and fragmented neutrophils. Wide-spread endothelial and liver Kupffer cell uptake followed intravenous administration. Human peripheral blood cells B cells, neutrophils, monocytes, and myeloid dendritic cells all efficiently bound Spike protein. Exposure to the Spike protein enhanced neutrophil NETosis and augmented human macrophage TNF-α and IL-6 production. Human and murine immune cells employed C-type lectin receptors and Siglecs to help capture the Spike protein. This study highlights the potential toxicity of the SARS-CoV-2 Spike protein for mammalian cells and illustrates the central role for alveolar macrophage in pathogenic protein uptake.

MAdCAM-1 costimulation in the presence of retinoic acid and TGF-ß promotes HIV infection and differentiation of CD4+ T cells into CCR5+ TRM-like cells.

CD4+ tissue resident memory T cells (TRMs) are implicated in the formation of persistent HIV reservoirs that are established during the very early stages of infection. The tissue-specific factors that direct T cells to establish tissue residency are not well defined, nor are the factors that establish viral latency. We report that costimulation via MAdCAM-1 and retinoic acid (RA), two constituents of gut tissues, together with TGF-ß, promote the differentiation of CD4+ T cells into a distinct subset α4ß7+CD69+CD103+ TRM-like cells. Among the costimulatory ligands we evaluated, MAdCAM-1 was unique in its capacity to upregulate both CCR5 and CCR9. MAdCAM-1 costimulation rendered cells susceptible to HIV infection. Differentiation of TRM-like cells was reduced by MAdCAM-1 antagonists developed to treat inflammatory bowel diseases. These finding provide a framework to better understand the contribution of CD4+ TRMs to persistent viral reservoirs and HIV pathogenesis.

Differential Effect of Extracellular Vesicles Derived from Plasmodium falciparum-Infected Red Blood Cells on Monocyte Polarization.

Malaria is a life-threatening tropical arthropod-borne disease caused by Plasmodium spp. Monocytes are the primary immune cells to eliminate malaria-infected red blood cells. Thus, the monocyte's functions are one of the crucial factors in controlling parasite growth. It is reasoned that the activation or modulation of monocyte function by parasite products might dictate the rate of disease progression. Extracellular vesicles (EVs), microvesicles, and exosomes, released from infected red blood cells, mediate intercellular communication and control the recipient cell function. This study aimed to investigate the physical characteristics of EVs derived from culture-adapted P. falciparum isolates (Pf-EVs) from different clinical malaria outcomes and their impact on monocyte polarization. The results showed that all P. falciparum strains released similar amounts of EVs with some variation in size characteristics. The effect of Pf-EV stimulation on M1/M2 monocyte polarization revealed a more pronounced effect on CD14+CD16+ intermediate monocytes than the CD14+CD16- classical monocytes with a marked induction of Pf-EVs from a severe malaria strain. However, no difference in the levels of microRNAs (miR), miR-451a, miR-486, and miR-92a among Pf-EVs derived from virulent and nonvirulent strains was found, suggesting that miR in Pf-EVs might not be a significant factor in driving M2-like monocyte polarization. Future studies on other biomolecules in Pf-EVs derived from the P. falciparum strain with high virulence that induce M2-like polarization are therefore recommended.

Extracellular Vesicles Derived from Early and Late Stage Plasmodium falciparum-Infected Red Blood Cells Contain Invasion-Associated Proteins.

In infectious diseases, extracellular vesicles (EVs) released from a pathogen or pathogen-infected cells can transfer pathogen-derived biomolecules, especially proteins, to target cells and consequently regulate these target cells. For example, malaria is an important tropical infectious disease caused by Plasmodium spp. Previous studies have identified the roles of Plasmodium falciparum-infected red blood cell-derived EVs (Pf-EVs) in the pathogenesis, activation, and modulation of host immune responses. This study investigated the proteomic profiles of Pf-EVs isolated from four P. falciparum strains. We also compared the proteomes of EVs from (i) different EV types (microvesicles and exosomes) and (ii) different parasite growth stages (early- and late-stage). The proteomic analyses revealed that the human proteins carried in the Pf-EVs were specific to the type of Pf-EVs. By contrast, most of the P. falciparum proteins carried in Pf-EVs were common across all types of Pf-EVs. As the proteomics results revealed that Pf-EVs contained invasion-associated proteins, the effect of Pf-EVs on parasite invasion was also investigated. Surprisingly, the attenuation of parasite invasion efficiency was found with the addition of Pf-MVs. Moreover, this effect was markedly increased in culture-adapted isolates compared with laboratory reference strains. Our evidence supports the concept that Pf-EVs play a role in quorum sensing, which leads to parasite growth-density regulation.

Enumeration of the Invasion Efficiency of Plasmodium falciparum In Vitro in Four Different Red Blood Cell Populations Using a Three-Color Flow Cytometry-Based Method.

A novel in vitro culture system using variable concentrations of biotin/streptavidin to label red blood cells (RBCs) that allows for the simultaneous comparison of growth rates in Plasmodium falciparum malaria parasite in four heterogeneous target RBC populations is described. Donor RBCs containing both P. falciparum-infected RBCs and non-infected RBCs at 0.5% parasitemia were first labeled with 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) succinimidyl ester (DDAO-SE) followed by co-culture with a mixture of equal numbers of four differentially biotin/streptavidin labeled RBC populations. After two to three schizogonic growth cycles, co-cultures were harvested and stained with streptavidin-phycoerythrin (SA-PE) followed by staining of parasite-infected RBCs with nucleic acid fluorochrome SYBR Green I. To demonstrate the application of this method, some target RBC populations that had sialic acid residues removed using neuraminidase treatment were mixed with RBC populations without enzymatic treatment and incubated with donor parasitized RBCs strain W2 (sialic acid-dependent) or 3D7 (sialic acid-independent). Significant less susceptibility to malaria parasite invasion was obtained with enzyme-treated RBC populations when compared with non-treated RBCs in blood samples from the same individual when using malaria parasite strain W2, whereas no difference in percent parasitemias was noted following infection with malaria parasite strain 3D7. This novel malaria culture method is cheap and provides increased sensitivity for direct comparison of parasite growth over time of any of the four RBC populations under identical conditions and eliminates the experimental bias due to contaminated donor RBCs. The application of biotin-labeled RBCs will therefore provide a better understanding of invasion phenotype-specific host-parasite interactions and the extent of complex malaria invasion mechanism. © 2019 International Society for Advancement of Cytometry.

Normalized levels of red blood cells expressing phosphatidylserine, their microparticles, and activated platelets in young patients with ß-thalassemia following bone marrow transplantation.

Bone marrow transplantation (BMT) serves as the only curative treatment for patients with ß-thalassemia major; however, hemostatic changes have been observed in these BMT patients. Aggregability of thalassemic red blood cells (RBCs) and increased red blood cell-derived microparticles (RMPs) expressing phosphatidylserine (PS) are thought to participate in thromboembolic events by initially triggering platelet activation. To our knowledge, there has been no report providing quantitation of these circulating PS-expressing RBCs and RMPs in young ß-thalassemia patients after BMT. Whole blood from each subject was fluorescently labeled to detect RBC markers (CD235a) and annexin-V together with the known number TruCount™ beads. PS-expressing RBCs, RMPs, and activated platelets were identified by flow cytometry. In our randomized study, we found the decreased levels of three aforementioned factors compared to levels in patients receiving regular blood transfusion (RT). This study showed that BMT in ß-thalassemia patients decreases the levels of circulating PS-expressing RBCs, their MPs, and procoagulant platelets when compared to patients who received RT. Normalized levels of these coagulation markers may provide the supportive evidence of the effectiveness of BMT for curing thalassemia.