Hypercholesterolemia exacerbates virus-induced immunopathologic liver disease via suppression of antiviral cytotoxic T cell responses

The immune system has to be optimally balanced to be highly effective against infections with cytopathic microbial pathogens and must guarantee efficient destruction of cells infected with noncytopathic agents while leaving the integrity of noninfected cells largely unaltered. We describe here the effects of genetically induced hypercholesterolemia on cellular immunity in apolipoprotein E (ApoE(-/-)) and low density lipoprotein receptor-deficient (LDLR(-/-)) mice during infection with the hepatotropic lymphocytic choriomeningitis virus WE strain. In both ApoE(-/-) and LDLR(-/-) mice hypercholesterolemia aggravated virus-induced immunopathologic liver disease. ApoE(-/-) mice exhibited a higher susceptibility to virus-induced immunopathology than LDLR(-/-) mice and usually succumbed to immunopathologic disease when infected with high doses of virus. Initial virus spread was not influenced by the hypercholesterolemia, whereas clearance of the virus from spleen and nonlymphoid organs, including liver, was delayed. Activation of antiviral CTL, measured by ex vivo cytotoxicity and IFN-gamma production, and recruitment of specific CTL into blood and liver were impaired in hypercholesterolemic mice, indicating that hypercholesterolemia had a significant suppressive effect on cellular immunity. Taken together, these data provide evidence that hypercholesterolemia suppresses antiviral immune responses, thereby changing the host-virus balance, and can increase susceptibility to acute or chronic and potentially lethal virus-induced immunopathologic disease. These findings impinge on our understanding of hypercholesterolemia as a disease parameter and may explain aspects of the frequent association of persistent pathogens with hypercholesterolemia-induced diseases, such as atherosclerosis.     

Rapid peptide turnover and inefficient presentation of exogenous antigen critically limit the activation of self-reactive CTL by dendritic cells

This study evaluated to what extent presentation of exogenously acquired self-Ags via MHC class I molecules on DC might contribute to the activation of self-reactive CTL and subsequent development of autoimmune disease. We show here by using the rat insulin promotor lymphocytic choriomeningitis virus glycoprotein model of autoimmune diabetes that the activation of self-reactive CTL by DC after uptake of exogenous Ag is very limited, first by the short half-life of MHC class I-associated peptides on DC in vitro and in vivo, and second by the rather inefficient MHC class I presentation of cell-associated self-Ags by DC. These two mechanisms are probably crucial in establishing high thresholds for the induction of self-reactive CTL that prevent autoimmune sequelae after release of sequestered and previously immunologically ignored tissue Ags.     

Marginal zone macrophages and immune responses against viruses

The effective establishment of antiviral protection requires a coordinated interplay between the innate and adaptive immune system. Using osteopetrotic (op(-/-)) mice, this study investigated the influence of marginal zone macrophages in controlling and initiating a protective immune response against a cytopathic vs a non- or low-cytopathic virus. Despite the generation of potent adaptive immune responses, antiviral protection against cytopathic vesicular stomatitis virus critically depended on the presence of marginal zone macrophages. Infection with low doses (100 PFU) of non- or low-cytopathic lymphocytic choriomeningitis virus was rarely cleared and usually resulted in a carrier state in the majority of mice. This shows that the early innate immune system provides an important preparatory phase to the adaptive immune system and is particularly important for antiviral protection.     

Correlation of T cell independence of antibody responses with antigen dose reaching secondary lymphoid organs: implications for splenectomized patients and vaccine design

Many natural viral and bacterial pathogens activate B cells independently of Th cells (TI Ags). This study analyzed the characteristics of the activation of B cells after immunization with various forms of viral Ags using different immunization routes and found a decreasing dependence on T help with increasing amounts of Ag recruited to the spleen. Repetitive antigenic structure facilitated TI B cell responses if Ag was present in lymphoid organs. These results suggest that 1) Ag dose and localization in secondary lymphoid organs are the key for B cell activation in the absence of T help; 2) early TI Ab responses are crucial to protect against systemically spreading acute cytopathic infectious agents; and 3) there may be new rationales for improved vaccine design.     

FTY720 immunosuppression impairs effector T cell peripheral homing without affecting induction, expansion, and memory

FTY720 (2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol hydrochloride) prolongs survival of solid organ allografts in animal models. Mechanisms of FTY720 immunomodulation were studied in mice infected with lymphocytic choriomeningitis virus (LCMV) to assess T cell responses or with vesicular stomatitis virus to evaluate Ab responses. Oral FTY720 (0.3 mg/kg/day) did not affect LCMV replication and specific CTL and B cells were induced and expanded normally. Moreover, the anti-viral humoral immune responses were normal. However, FTY720 treatment showed first a shift of overall distribution of CTL from the spleen to peripheral lymph nodes and lymphocytopenia was observed. This effect was reversible within 7-21 days. Together with unimpaired T and B cell memory after FTY720 treatment, this finding rendered enhancement of lymphocyte apoptosis by FTY720 in vivo unlikely. Secondly, the delayed-type hypersensitivity reaction to a viral MHC class I-presented peptide was markedly reduced by FTY720. These results were supported by impaired circulation of LCMV specific TCR transgenic effector lymphocytes in the peripheral blood and reduced numbers of tissue infiltrating CTL in response to delayed-type hypersensitivity reaction. Thirdly, in a CD8+ T cell-mediated diabetes model in a transgenic mouse expressing the LCMV glycoprotein in the islets of the pancreas, FTY720 delayed or prevented disease by reducing islet-infiltrating CTL. Thus, FTY720 effectively reduced recirculation of CD8+ effector T cells and their recruitment to peripheral lesions without affecting the induction and expansion of immune responses in secondary lymphoid organs. These properties may offer the potential to treat ongoing organ-specific T cell-mediated immunopathologic disease.     

Recombinant renewable polyclonal antibodies

Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.     

Comparison of different delivery systems of DNA vaccination for the induction of protection against tuberculosis in mice and guinea pigs

The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly (DL-lactide-co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in mice as well as in guinea pigs by comparison with the efficacy and toxicity induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in both mice and guinea pigs.     

Hyperinflammatory environment drives dysfunctional myeloid cell effector response to bacterial challenge in COVID-19

COVID-19 displays diverse disease severities and symptoms including acute systemic inflammation and hypercytokinemia, with subsequent dysregulation of immune cells. Bacterial superinfections in COVID-19 can further complicate the disease course and are associated with increased mortality. However, there is limited understanding of how SARS-CoV-2 pathogenesis and hypercytokinemia impede the innate immune function against bacterial superinfections. We assessed the influence of COVID-19 plasma hypercytokinemia on the functional responses of myeloid immune cells upon bacterial challenges from acute-phase COVID-19 patients and their corresponding recovery-phase. We show that a severe hypercytokinemia status in COVID-19 patients correlates with the development of bacterial superinfections. Neutrophils and monocytes derived from COVID-19 patients in their acute-phase showed an impaired intracellular microbicidal capacity upon bacterial challenges. The impaired microbicidal capacity was reflected by abrogated MPO and reduced NETs production in neutrophils along with reduced ROS production in both neutrophils and monocytes. Moreover, we observed a distinct pattern of cell surface receptor expression on both neutrophils and monocytes, in line with suppressed autocrine and paracrine cytokine signaling. This phenotype was characterized by a high expression of CD66b, CXCR4 and low expression of CXCR1, CXCR2 and CD15 in neutrophils and low expression of HLA-DR, CD86 and high expression of CD163 and CD11b in monocytes. Furthermore, the impaired antibacterial effector function was mediated by synergistic effect of the cytokines TNF-α, IFN-γ and IL-4. COVID-19 patients receiving dexamethasone showed a significant reduction of overall inflammatory markers in the plasma as well as exhibited an enhanced immune response towards bacterial challenge ex vivo. Finally, broad anti-inflammatory treatment was associated with a reduction in CRP, IL-6 levels as well as length of ICU stay and ventilation-days in critically ill COVID-19 patients. Our data provides insights into the transient functional dysregulation of myeloid immune cells against subsequent bacterial infections in COVID-19 patients and describe a beneficial role for the use of dexamethasone in these patients.