Pathological effects of SARS-CoV-2 on hematological and immunological cells: Alterations in count, morphology, and function.

The novel Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19 outbreak, spread rapidly and infected more than 140 million people with more than three million victims worldwide. The SARS-CoV-2 causes destructive changes in the immunological and hematological system of the host. These alterations appear to play a critical role in disease pathology and the emerging of clinical manifestations. In this review, we aimed to discuss the effect of COVID-19 on the count, function and morphology of immune and blood cells and the role of these changes in the pathophysiology of the disease. Knowledge of these changes may help with better management and treatment of COVID-19 patients.

Endothelial dysfunction contributes to severe COVID-19 in combination with dysregulated lymphocyte responses and cytokine networks.

The systemic processes involved in the manifestation of life-threatening COVID-19 and in disease recovery are still incompletely understood, despite investigations focusing on the dysregulation of immune responses after SARS-CoV-2 infection. To define hallmarks of severe COVID-19 in acute disease (n = 58) and in disease recovery in convalescent patients (n = 28) from Hannover Medical School, we used flow cytometry and proteomics data with unsupervised clustering analyses. In our observational study, we combined analyses of immune cells and cytokine/chemokine networks with endothelial activation and injury. ICU patients displayed an altered immune signature with prolonged lymphopenia but the expansion of granulocytes and plasmablasts along with activated and terminally differentiated T and NK cells and high levels of SARS-CoV-2-specific antibodies. The core signature of seven plasma proteins revealed a highly inflammatory microenvironment in addition to endothelial injury in severe COVID-19. Changes within this signature were associated with either disease progression or recovery. In summary, our data suggest that besides a strong inflammatory response, severe COVID-19 is driven by endothelial activation and barrier disruption, whereby recovery depends on the regeneration of the endothelial integrity.

Systemic and mucosal mobilization of granulocyte subsets during lentiviral infection.

Granulocytes mediate broad immunoprotection through phagocytosis, extracellular traps, release of cytotoxic granules, antibody effector functions and recruitment of other immune cells against pathogens. However, descriptions of granulocytes in HIV infection and mucosal tissues are limited. Our goal was to characterize granulocyte subsets in systemic, mucosal and lymphoid tissues during lentiviral infection using the rhesus macaque (RM) model. Mononuclear cells from jejunum, colon, cervix, vagina, lymph nodes, spleen, liver and whole blood from experimentally naïve and chronically SHIVsf162p3-infected RM were analysed by microscopy and polychromatic flow cytometry. Granulocytes were identified using phenotypes designed specifically for RM: eosinophils-CD45+  CD66+  CD49d+ ; neutrophils-CD45+  CD66+  CD14+ ; and basophils-CD45+  CD123+  FcRε+ . Nuclear visualization with DAPI staining and surface marker images by ImageStream (cytometry/microscopy) further confirmed granulocytic phenotypes. Flow cytometric data showed that all RM granulocytes expressed CD32 (FcRγII) but did not express CD16 (FcRγIII). Additionally, constitutive expression of CD64 (FcRγI) on neutrophils and FcRε on basophils indicates the differential expression of Fc receptors on granulocyte subsets. Granulocytic subsets in naïve whole blood ranged from 25·4% to 81·5% neutrophils, 0·59% to 13·3% eosinophils and 0·059% to 1·8% basophils. Interestingly, elevated frequencies of circulating neutrophils, colorectal neutrophils and colorectal eosinophils were all observed in chronic lentiviral disease. Conversely, circulating basophils, jejunal eosinophils, vaginal neutrophils and vaginal eosinophils of SHIVsf162p3-infected RM declined in frequency. Overall, our data suggest modulation of granulocytes in chronic lentiviral infection, most notably in the gastrointestinal mucosae where a significant inflammation and disruption occurs in lentivirus-induced disease. Furthermore, granulocytes may migrate to inflamed tissues during infection and could serve as targets of immunotherapeutic intervention.

Can Activation of NRF2 Be a Strategy against COVID-19?

Acute respiratory distress syndrome (ARDS) caused by SARS-CoV-2 is largely the result of a dysregulated host response, followed by damage to alveolar cells and lung fibrosis. Exacerbated proinflammatory cytokines release (cytokine storm) and loss of T lymphocytes (leukopenia) characterize the most aggressive presentation. We propose that a multifaceted anti-inflammatory strategy based on pharmacological activation of nuclear factor erythroid 2 p45-related factor 2 (NRF2) can be deployed against the virus. The strategy provides robust cytoprotection by restoring redox and protein homeostasis, promoting resolution of inflammation, and facilitating repair. NRF2 activators such as sulforaphane and bardoxolone methyl are already in clinical trials. The safety and efficacy information of these modulators in humans, together with their well-documented cytoprotective and anti-inflammatory effects in preclinical models, highlight the potential of this armamentarium for deployment to the battlefield against COVID-19.

Prevalence of HHV-6 in endometrium from women with recurrent implantation failure.

PROBLEM: To study the prevalence of HHV-6 in endometrial biopsies among women experiencing recurrent implantation failure (RIF) after IVF/ET compared with controls. METHOD OF STUDY: Thirty women experiencing RIF after IVF/ET and 10 fertile women participated in the study. All women had endometrial biopsies taken in the luteal phase of their menstrual cycle for an endometrial immune profile (EIP) and HHV-6 mRNA as well as lymphocyte and granulocyte populations. The prevalence of HHV-6 in endometrial biopsies was determined, and biopsies for positive and negative expression of HHV-6 were compared with the results of their EIP and lymphocyte and granulocyte populations. RESULTS: Thirty-seven percentage of women with a history of RIF and 0% of controls demonstrated the presence of HHV-6 in their endometrial biopsies. No associations were found when the results of the endometrial immune profile were compared with the presence or absence of HHV-6. Significant increase in neutrophil-specific CD16b mRNA was found in HHV-6-positive samples, and the levels of B cells-related CD19 mRNA were lower in biopsies from women with RIF in comparison with normal controls. CONCLUSION: HHV-6 infection is an important factor in RIF.

Immune roles of amphibian (Xenopus laevis) tadpole granulocytes during Frog Virus 3 ranavirus infections.

Infections by Frog Virus 3 (FV3) and other ranaviruses (RVs) are contributing to the amphibian declines, while the mechanisms controlling anuran tadpole susceptibility and adult frog resistance to RVs, including the roles of polymorphonuclear granulocytes (PMNs) during anti-FV3 responses, remain largely unknown. Since amphibian kidneys represent an important FV3 target, the inability of amphibian (Xenopus laevis) tadpoles to mount effective kidney inflammatory responses to FV3 is thought to contribute to their susceptibility. Here we demonstrate that a recombinant X. laevis granulocyte colony-stimulating factor (G-CSF) generates PMNs with hallmark granulocyte morphology. Tadpole pretreatment with G-CSF prior to FV3 infection reduces animal kidney FV3 loads and extends their survival. Moreover, G-CSF-derived PMNs are resistant to FV3 infection and express high levels of TNFα in response to this virus. Notably, FV3-infected tadpoles fail to recruit G-CSFR expressing granulocytes into their kidneys, suggesting that they lack an integral inflammatory effector population at this site.

Deletion of Irf3 and Irf7 Genes in Mice Results in Altered Interferon Pathway Activation and Granulocyte-Dominated Inflammatory Responses to Influenza A Infection.

The interferon (IFN) pathway plays an essential role in the innate immune response following viral infections and subsequent shaping of adaptive immunity. Infections with influenza A viruses (IAV) activate the IFN pathway after the recognition of pathogen-specific molecular patterns by respective pattern recognition receptors. The IFN regulatory factors IRF3 and IRF7 are key players in the regulation of type I and III IFN genes. In this study, we analyzed the role of IRF3 and IRF7 for the host response to IAV infections in Irf3-/-, Irf7-/-, and Irf3-/-Irf7-/- knockout mice. While the absence of IRF3 had only a moderate impact on IFN expression, deletion of IRF7 completely abolished IFNα production after infection. In contrast, lack of both IRF3 and IRF7 resulted in the absence of both IFNα and IFNß after IAV infection. In addition, IAV infection of double knockout mice resulted in a strong increase of mortality associated with a massive influx of granulocytes in the lung and reduced activation of the adaptive immune response.

HIV infection: focus on the innate immune cells.

Innate immune cells play a critical role during the onset of HIV infection and remain active until the final events that characterize AIDS. The viral impact on innate immune cell response may be a result of direct infection or indirect modulation, and each cell type responds in a specific manner to HIV. During HIV infection, the immune system works in a dynamic way, where innate and adaptive cells contribute with each other stimulating their function and modulating phenotypes and consequently infection resolution. Understanding the alterations in the cell populations induced by the virus is pivotal and can help to combat HIV at the time of infection and above all, to prevent the establishment of viral reservoirs. In this review, we will describe the frequency and the subtypes of infected cells such as of monocytes, DCs, neutrophils, eosinophils, mast cells/basophils, NK cells, NKT cells and γδ T cells, and we discuss the possibility of cell-targeting strategies. Our aim is to consolidate the existing knowledge of the interaction between HIV and cells that constitute the innate immune response.

High expression levels of BLyS/BAFF by blood dendritic cells and granulocytes are associated with B-cell dysregulation in SIV-infected rhesus macaques.

Dendritic cells (DCs) modulate B-cell survival and differentiation, mainly through production of growth factors such as B lymphocyte stimulator (BLyS/BAFF). In recent longitudinal studies involving HIV-1-infected individuals with different rates of disease progression, we have shown that DCs were altered in number and phenotype in the context of HIV-1 disease progression and B-cell dysregulations were associated with increased BLyS/BAFF expression in plasma and by blood myeloid DCs (mDCs) in rapid and classic progressors but not in HIV-1-elite controllers (EC). Suggesting that the extent to which HIV-1 disease progression is controlled may be linked to BLyS/BAFF expression status and the capacity to orchestrate B-cell responses. Herein, longitudinal analyses of simian immunodeficiency virus (SIV)-infected rhesus macaques also revealed increased expression of BLyS/BAFF by blood mDCs as soon as day 8 and throughout infection. Strikingly, granulocytes presented the highest BLyS/BAFF expression profile in the blood of SIV-infected macaques. BLyS/BAFF levels were also increased in plasma and correlated with viral loads. Consequently, these SIV-infected animals had plasma hyperglobulinemia and reduced blood B-cell numbers with altered population frequencies. These data underscore that BLyS/BAFF is associated with immune dysregulation in SIV-infected rhesus macaques and suggest that BLyS/BAFF is a key regulator of immune activation that is highly conserved among primates. These findings emphasize the potential importance of this SIV-infected primate model to test whether blocking excess BLyS/BAFF has an effect on the overall inflammatory burden and immune restoration.

Interferon at the crossroads of allergy and viral infections.

IFN-α/ß was first described as a potent inhibitor of viral replication, but it is now appreciated that IFN signaling plays a pleiotropic role in regulating peripheral T cell functions. Recently, IFN-α/ß was shown to block human Th2 development by suppressing the transcription factor GATA3. This effect is consistent with the role for IFN-α/ß in suppressing allergic inflammatory processes by blocking granulocyte activation and IL-4-mediated B cell isotype switching to IgE. With the consideration of recent studies demonstrating a defect in IFN-α/ß secretion in DCs and epithelial cells from individuals with severe atopic diseases, there is an apparent reciprocal negative regulatory loop in atopic individuals, whereby the lack of IFN-α/ß secretion by innate cells contributes to the development of allergic Th2 cells. Is it possible to overcome these events by treating with IFN-α/ß or by inducing its secretion in vivo? In support of this approach, case studies have documented the therapeutic potential of IFN-α/ß in treating steroid-resistant allergic asthma and other atopic diseases. Additionally, individuals with asthma who are infected with HCV and respond to IFN therapy showed a reduction in symptoms and severity of asthma attacks. These findings support a model, whereby allergic and antiviral responses are able to cross-regulate each other, as IgER cross-linking of pDCs prevents IFN-α/ß production in response to viral infection. The clinical importance of upper-respiratory viruses in the context of allergic asthma supports the need to understand how these pathways intersect and to identify potential therapeutic targets.

Quantitative and ultrastructural changes in the haemocytes of Spodoptera littoralis (Boisd.) treated individually or in combination with Spodoptera littoralis multicapsid nucleopolyhedrovirus (SpliMNPV) and azadirachtin.

The total haemocyte count (THC) and the possible ultrastructural alterations induced in the haemocytes of the fourth larval instars of the Egyptian cotton leafworm, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae), 96 h post-feeding on a semi-synthetic diet, treated with the LC50 of Spodoptera littoralis multicapsid nucleopolyhedrovirus (SpliMNPV) and the LC50 of azadirachtin alone, and the LC25 of SpliMNPV combined with the LC25 of azadirachtin were studied and compared to the control. Single treatment with the virus and azadirachtin or combined treatment significantly decreased the THC compared to the control. There are five types of haemocytes in S. littoralis: prohaemocytes, plasmatocytes, granulocytes, spherulocytes and oenocytoids. The most common symptoms in granulocytes and plasmatocytes, the main affected cell types, due to viral infection were the presence of virogenic stroma, peripheral dispersion of the chromatin and disappearance of the nucleoli. However, the most common symptoms in these two types of haemocytes due to treatment with azadirachtin were the presence of rough endoplasmic reticulum filled with fibrous materials, due to probably apoptosis, in their cisternae and disorganization of mitochondria (looped, vacuolated and swollen). In addition, the cytoplasm of granulocytes was vacuolated with the appearance of autophagic lysosomes, while plasmatocytes showed ruptured cell membrane and folded nuclear envelope. Combined treatment with the NPV and azadirachtin induced the same pathological changes which were recorded from individual treatment with the virus or azadirachtin to the same haemocytes. It can be concluded that the change in the THC and ultrastructure of granulocytes and plasmatocytes may affect the cellular-mediated immune response in S. littoralis. Moreover, it seems likely that mitochondria were the target site of azadirachtin, as they were affected in both granulocytes and plasmatocytes treated with azadirachtin alone or in combination with SpliMNPV.

Granulocytes of the red claw crayfish Cherax quadricarinatus can endocytose beads, E. coli and WSSV, but in different ways.

The hemocytes of the red claw crayfish Cherax quadricarinatus are classified by morphologic observation into the following types: hyalinocytes (H), semi-granulocytes (SG) and granulocytes (G). Density gradient centrifugation with Percoll was developed to separate these three subpopulations of hemocytes. Beads, Escherichia coli, and FITC labeling WSSV were used to investigate the characteristics of granulocytes by using scanning electron microscope, transmission electron microscope, and laser scan confocal microscope. Results showed that granulocytes could phagocytose beads and E. coli by endocytic pathways. WSSV could rely on caveolae-mediated endocytosis to mainly enter into granulocytes. These results could elucidate the mechanism of the innate immunity function of granulocytes, and it also showed the mechanism by which WSSV invaded granulocytes in the red claw crayfish.

Development of a FACS-based assay for evaluating antiviral potency of compound in dengue infected peripheral blood mononuclear cells.

Dengue fever is the most important arthropod-transmitted viral disease affecting humans. It is a blood-borne disease characterized by persistent fever and joint pain. In the blood, primary peripheral blood mononuclear cells (PBMCs), in particular monocytes, are the main target of the dengue virus (DENV). These cells are poorly permissive for in vitro dengue virus infection and their infectivity varies from donor to donor. To overcome this barrier, an anti-dengue antibody was used to improve the infectivity of DENV-2 clinical isolates to PBMCs, the monocytic leukemia cell line, THP-1 and the granulocyte cell line, KU812. A higher throughput 96-well-format assay based on a fluorescent-activated cell sorter could potentially be developed to evaluate the antiviral potency of compounds in DENV-infected PBMCs in vitro. The results correlate well with data obtained by a standard plaque assay. Altogether, an assay has been developed that enables evaluation of the antiviral activity of test compounds in a physiologically-relevant cell system (PBMCs). These screening processes are urgently needed for dengue drug discovery.

Nonreplicating vaccines can protect african green monkeys from the memphis 37 strain of respiratory syncytial virus.

BACKGROUND: We evaluated the immunological responses of African green monkeys immunized with multiple F and G protein-based vaccines and assessed protection against the Memphis 37 strain of respiratory syncytial virus (RSV). METHODS: Monkeys were immunized with F and G proteins adjuvanted with immunostimulatory (CpG) oligodeoxyribonucleotides admixed with either Alhydrogel or ISCOMATRIX adjuvant. Delivery of F and G proteins via replication incompetent recombinant vesicular stomatitis viruses (VSVs) and human adenoviruses was also evaluated. Mucosally or parenterally administered recombinant adenoviruses were used in prime-boost regimens with adjuvanted proteins or recombinant DNA. RESULTS: Animals primed by intranasal delivery of recombinant adenoviruses, and boosted by intramuscular injection of adjuvanted F and G proteins, developed neutralizing antibodies and F/G protein-specific T cells and were protected from RSV infection. Intramuscular injections of Alhydrogel (plus CpG) adjuvanted F and G proteins reduced peak viral loads in the lungs of challenged monkeys. Granulocyte numbers were not significantly elevated, relative to controls, in postchallenge bronchoalveolar lavage samples from vaccinated animals. CONCLUSIONS: This study has validated the use of RSV (Memphis 37) in an African green monkey model of intranasal infection and identified nonreplicating vaccines capable of eliciting protection in this higher species challenge model.

An infectious HHV-6B isolate from a healthy adult with chromosomally integrated virus and a reporter based relative viral titer assay.

Human herpesvirus 6B (HHV-6B) primary infections occur in early childhood and establish a life-long latency in the most healthy adults. HHV-6B was detectable in the peripheral blood mononuclear cells (PBMC) and granulocytes by serial genomic DNA dilution PCR till 10 pg of template DNA, in a healthy adult. Epstein Barr virus (EBV) mediated transformation of the PBMC resulted in establishment of a B-cell line. Southern hybridization with the PBMC as well as the cell line DNA showed distinct signals for high copy viral genomes and Gardella gel analysis indicated chromosomal integration of the HHV-6B. Integration site analysis in the PBMC and the cell line indicated an atypical viral integration in non-telomeric region of chromosome 12. Cell free culture medium of the cell line could infect different mononuclear cell lines, naïve or mitogen stimulated PBMC and was found to impart productive infection in a recipient T cell line. An HIV-1 LTR driven luciferase based reporter cell line was made and a single step assay was developed for estimating HHV-6B relative concentration in the culture supernatants. This study thus reports a new infectious HHV-6B isolate with uncommon integration site, spontaneous production from a cell line and also development of a simple relative HHV-6B titer assay.

Apoptosis of peripheral blood leucocytes in rabbits infected with different strains of rabbit haemorrhagic disease virus.

The pathogenicity of RHDV (rabbit haemorrhagic disease virus) is mainly associated with its affinity to blood vessels, with causing disseminated intravascular coagulations (DIC), and with the stimulation of the host immune system. Moreover, there are implications suggesting that apoptosis may be a pivotal process in understanding the basis of viral haemorrhagic disease in rabbits - a serious infectious disease causing mortality to wild and domestic rabbits. The aim of this study is to evaluate, by means of flow cytometry, the dynamics of apoptosis in peripheral blood granulocytes and lymphocytes in rabbits experimentally infected with seven different strains of RHDV and so-called antigenic variants of RHDV denominated as RHDVa, i.e.: Hungarian 24V/89, 1447V/96, 72V/2003; Austrian 01-04, 237/04, V-412 and French 05-01. The results showed that all of the RHDV and RHDVa strains cause an increase in the number of apoptotic cells throughout the infection, which might indicate the need for further analysis of the importance of this process.

The role of zinc on anti-Newcastle disease virus specific antibody response and agranulocytes count in rabbits treated with methotrexate and prednisolone.

Zinc (Zn) plays a pivotal role in highly proliferative tissues including immune system. The long-term therapy of neoplastic and autoimmune disorders is associated with immunosuppression and myleosuppression. In the current study role of Zn on anti-Newcastle disease virus response and agranulocytes count of methotrexate and prednisolone treated rabbits. Thirty six healthy rabbits were randomly segregated into six groups (group I to VI) each containing six rabbits. Oil based Newcastle disease virus (NDV) vaccine was administered subcutaneously to rabbits of all the groups at day 0 and 21 and after one week, all the groups received Zn, (Zn + prednisolone), prednisolone, (Zn + methotrexate) methotrexate orally from day 7 to day 21, except the control. The serum antibody titer, total and differential leukocyte count were measured weekly for 6 weeks. The administration of zinc in combination with methotrexate showed same antibody titer as that of the control suggesting that Zn has ability to counteract the methotrexate-induced immunosuppression. However, Zn did not show any significant impact in combination with prednisolone (p<0.05). The results of the present study indicate that co-administration of Zn and methotrexate is beneficial in the activity of immune system.

Cell carriage, delivery, and selective replication of an oncolytic virus in tumor in patients.

Oncolytic viruses, which preferentially lyse cancer cells and stimulate an antitumor immune response, represent a promising approach to the treatment of cancer. However, how they evade the antiviral immune response and their selective delivery to, and replication in, tumor over normal tissue has not been investigated in humans. Here, we treated patients with a single cycle of intravenous reovirus before planned surgery to resect colorectal cancer metastases in the liver. Tracking the viral genome in the circulation showed that reovirus could be detected in plasma and blood mononuclear, granulocyte, and platelet cell compartments after infusion. Despite the presence of neutralizing antibodies before viral infusion in all patients, replication-competent reovirus that retained cytotoxicity was recovered from blood cells but not plasma, suggesting that transport by cells could protect virus for potential delivery to tumors. Analysis of surgical specimens demonstrated greater, preferential expression of reovirus protein in malignant cells compared to either tumor stroma or surrounding normal liver tissue. There was evidence of viral factories within tumor, and recovery of replicating virus from tumor (but not normal liver) was achieved in all four patients from whom fresh tissue was available. Hence, reovirus could be protected from neutralizing antibodies after systemic administration by immune cell carriage, which delivered reovirus to tumor. These findings suggest new preclinical and clinical scheduling and treatment combination strategies to enhance in vivo immune evasion and effective intravenous delivery of oncolytic viruses to patients in vivo.

HIV-1 Tat-induced microgliosis and synaptic damage via interactions between peripheral and central myeloid cells.

Despite the ability of combination antiretroviral treatment (cART) to reduce viral burden to nearly undetectable levels in cerebrospinal fluid and serum, HIV-1 associated neurocognitive disorders (HAND) continue to persist in as many as half the patients living with this disease. There is growing consensus that the actual substrate for HAND is destruction of normal synaptic architecture but the sequence of cellular events that leads to this outcome has never been resolved. To address whether central vs. peripheral myeloid lineage cells contribute to synaptic damage during acute neuroinflammation we injected a single dose of the HIV-1 transactivator of transcription protein (Tat) or control vehicle into hippocampus of wild-type or chimeric C57Bl/6 mice genetically marked to distinguish infiltrating and resident immune cells. Between 8-24 hr after injection of Tat, invading CD11b(+) and/or myeloperoxidase-positive leukocytes with granulocyte characteristics were found to engulf both microglia and synaptic structures, and microglia reciprocally engulfed invading leukocytes. By 24 hr, microglial processes were also seen ensheathing dendrites, followed by inclusion of synaptic elements in microglia 7 d after Tat injection, with a durable microgliosis lasting at least 28 d. Thus, central nervous system (CNS) exposure to Tat induces early activation of peripheral myeloid lineage cells with phagocytosis of synaptic elements and reciprocal microglial engulfment of peripheral leukocytes, and enduring microgliosis. Our data suggest that a single exposure to a foreign antigen such as HIV-1 Tat can lead to long-lasting disruption of normal neuroimmune homeostasis with deleterious consequences for synaptic architecture, and further suggest a possible mechanism for enduring neuroinflammation in the absence of productive viral replication in the CNS.