Reduced cardiovascular risk score following bilateral cataract phacoemulsification surgery: A retrospective observational cohort study.

OBJECTIVE: To assess the cardiovascular risk (CV risk) change following bilateral phacoemulsification cataract surgery. METHODS: We performed a retrospective observation cohort study on 112 selected patients who underwent uncomplicated bilateral cataract surgery at Centro Hospitalar de Entre o Douro e Vouga (CHEDV) between 2018 and 2019. This patient cohort was further subdivided in 2 different groups: Good VA - no to mild visual impairment, ≤0.48 LogMAR; Bad VA - moderate to severe visual impairment, >0.48 LogMAR. We compared the changes in the CV risk score components in our patient cohort and between subgroups Good VA and Bad VA, before and after surgery, using paired t-test or Wilcoxon rank-sum test, and repeated measures ANOVA with Tukey post-hoc tests, respectively. Visual Acuity (VA) before and after surgery was correlated with the patients' CV risk score. At last, linear regression models were built to explain changes in CV risk variables considering the change in VA. RESULTS: Cataract surgery resulted in improved VA. Notably, following surgery our patient cohort showed reduced low-density lipoprotein (LDL) levels after surgery, from 111.17±36.26 mg/dL to 104.22±37.53 mg/dL, and reduced systolic arterial pressure (SAP), from 139.1±15.0 mmHg to 133.7±12.0 mmHg. Ultimately, this translated to an improved CV risk score within 6 months of cataract surgery, from 17.39±11.44% to 16.51±11.27%. Of note, these improvements were mostly present in the Bad VA group of patients, where baseline VA and incidence of dyslipidemia were worse. CONCLUSION: Our results suggest that phacoemulsification cataract surgery may be an important tool in addressing CV risk.

Mechanisms of Antiviral Cytotoxic CD4 T Cell Differentiation.

Cytotoxic CD4 T lymphocytes (CD4-CTL) are important in antiviral immunity. For example, we have previously shown that in mice, CD4-CTL are important to control ectromelia virus (ECTV) infection. How viral infections induce CD4-CTL responses remains incompletely understood. We demonstrate here that not only ECTV but also vaccinia virus and lymphocytic choriomeningitis virus induce CD4-CTL, though the response to ECTV is stronger. Using ECTV, we also demonstrate that in contrast to CD8-CTL, CD4-CTL differentiation requires constant virus replication and ceases once the virus is controlled. We also show that major histocompatibility complex class II molecules on CD11c+ cells are required for CD4-CTL differentiation and for mousepox resistance. Transcriptional analysis indicated that antiviral CD4-CTL and noncytolytic T helper 1 (Th1) CD4 T cells have similar transcriptional profiles, suggesting that CD4-CTL are terminally differentiated classical Th1 cells. Interestingly, CD4-CTL and classical Th1 cells expressed similar mRNA levels of the transcription factors ThPOK and GATA-3, necessary for CD4 T cell linage commitment, and Runx3, required for CD8 T cell development and effector function. However, at the protein level, CD4-CTL had higher levels of the three transcription factors, suggesting that further posttranscriptional regulation is required for CD4-CTL differentiation. Finally, CRISPR/Cas9-mediated deletion of Runx3 in CD4 T cells inhibited CD4-CTL but not classical Th1 cell differentiation in response to ECTV infection. These results further our understanding of the mechanisms of CD4-CTL differentiation during viral infection and the role of posttranscriptionally regulated Runx3 in this process. IMPORTANCE While it is well established that cytotoxic CD4 T cells (CD4-CTLs) directly contribute to viral clearance, it remains unclear how CD4-CTL are induced. We now show that CD4-CTLs require sustained antigen presentation and are induced by CD11c-expressing antigen-presenting cells. Moreover, we show that CD4-CTLs are derived from the terminal differentiation of classical T helper 1 (Th1) subset of CD4 cells. Compared to Th1 cells, CD4-CTLs upregulate protein levels of the transcription factors ThPOK, Runx3, and GATA-3 posttranscriptionally. Deletion of Runx3 in differentiated CD4 T cells prevents induction of CD4-CTLs but not classical Th1 cells. These results advance our knowledge of how CD4-CTLs are induced during viral infection.

Resistance to lethal ectromelia virus infection requires Type I interferon receptor in natural killer cells and monocytes but not in adaptive immune or parenchymal cells.

Type I interferons (IFN-I) are antiviral cytokines that signal through the ubiquitous IFN-I receptor (IFNAR). Following footpad infection with ectromelia virus (ECTV), a mouse-specific pathogen, C57BL/6 (B6) mice survive without disease, while B6 mice broadly deficient in IFNAR succumb rapidly. We now show that for survival to ECTV, only hematopoietic cells require IFNAR expression. Survival to ECTV specifically requires IFNAR in both natural killer (NK) cells and monocytes. However, intrinsic IFNAR signaling is not essential for adaptive immune cell responses or to directly protect non-hematopoietic cells such as hepatocytes, which are principal ECTV targets. Mechanistically, IFNAR-deficient NK cells have reduced cytolytic function, while lack of IFNAR in monocytes dampens IFN-I production and hastens virus dissemination. Thus, during a pathogenic viral infection, IFN-I coordinates innate immunity by stimulating monocytes in a positive feedback loop and by inducing NK cell cytolytic function.

Lipid-nanoparticle-encapsulated mRNA vaccines induce protective memory CD8 T cells against a lethal viral infection.

It is well established that memory CD8 T cells protect susceptible strains of mice from mousepox, a lethal viral disease caused by ectromelia virus (ECTV), the murine counterpart to human variola virus. While mRNA vaccines induce protective antibody (Ab) responses, it is unknown whether they also induce protective memory CD8 T cells. We now show that immunization with different doses of unmodified or N(1)-methylpseudouridine-modified mRNA (modified mRNA) in lipid nanoparticles (LNP) encoding the ECTV gene EVM158 induced similarly strong CD8 T cell responses to the epitope TSYKFESV, albeit unmodified mRNA-LNP had adverse effects at the inoculation site. A single immunization with 10 µg modified mRNA-LNP protected most susceptible mice from mousepox, and booster vaccination increased the memory CD8 T cell pool, providing full protection. Moreover, modified mRNA-LNP encoding TSYKFESV appended to green fluorescent protein (GFP) protected against wild-type ECTV infection while lymphocytic choriomeningitis virus glycoprotein (GP) modified mRNA-LNP protected against ECTV expressing GP epitopes. Thus, modified mRNA-LNP can be used to create protective CD8 T cell-based vaccines against viral infections.

Viral infection modulates Qa-1b in infected and bystander cells to properly direct NK cell killing.

Natural killer (NK) cell activation depends on the signaling balance of activating and inhibitory receptors. CD94 forms inhibitory receptors with NKG2A and activating receptors with NKG2E or NKG2C. We previously demonstrated that CD94-NKG2 on NK cells and its ligand Qa-1b are important for the resistance of C57BL/6 mice to lethal ectromelia virus (ECTV) infection. We now show that NKG2C or NKG2E deficiency does not increase susceptibility to lethal ECTV infection, but overexpression of Qa-1b in infected cells does. We also demonstrate that Qa-1b is down-regulated in infected and up-regulated in bystander inflammatory monocytes and B cells. Moreover, NK cells activated by ECTV infection kill Qa-1b-deficient cells in vitro and in vivo. Thus, during viral infection, recognition of Qa-1b by activating CD94/NKG2 receptors is not critical. Instead, the levels of Qa-1b expression are down-regulated in infected cells but increased in some bystander immune cells to respectively promote or inhibit their killing by activated NK cells.

α2ß1 Integrin Is Required for Optimal NK Cell Proliferation during Viral Infection but Not for Acquisition of Effector Functions or NK Cell-Mediated Virus Control.

NK cells play an important role in antiviral resistance. The integrin α2, which dimerizes with integrin ß1, distinguishes NK cells from innate lymphoid cells 1 and other leukocytes. Despite its use as an NK cell marker, little is known about the role of α2ß1 in NK cell biology. In this study, we show that in mice α2ß1 deficiency does not alter the balance of NK cell/ innate lymphoid cell 1 generation and slightly decreases the number of NK cells in the bone marrow and spleen without affecting NK cell maturation. NK cells deficient in α2ß1 had no impairment at entering or distributing within the draining lymph node of ectromelia virus (ECTV)-infected mice or at becoming effectors but proliferated poorly in response to ECTV and did not increase in numbers following infection with mouse CMV (MCMV). Still, α2ß1-deficient NK cells efficiently protected from lethal mousepox and controlled MCMV titers in the spleen. Thus, α2ß1 is required for optimal NK cell proliferation but is dispensable for protection against ECTV and MCMV, two well-established models of viral infection in which NK cells are known to be important.

Chronic Lymphocytic Choriomeningitis Infection Causes Susceptibility to Mousepox and Impairs Natural Killer Cell Maturation and Function.

Chronic viral infections. like those of humans with cytomegalovirus, human immunodeficiency virus (even when under antiretroviral therapy), and hepatitis C virus or those of mice with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), result in immune dysfunction that predisposes the host to severe infections with unrelated pathogens. It is known that C57BL/6 (B6) mice are resistant to mousepox, a lethal disease caused by the orthopoxvirus ectromelia virus (ECTV), and that this resistance requires natural killer (NK) cells and other immune cells. We show that most B6 mice chronically infected with CL13 succumb to mousepox but that most of those that recovered from acute infection with the LCMV Armstrong (Arm) strain survive. We also show that B6 mice chronically infected with CL13 and those that recovered from Arm infection have a reduced frequency and a reduced number of NK cells. However, at steady state, NK cells in mice that have recovered from Arm infection mature normally and, in response to ECTV, get activated, become more mature, proliferate, and increase their cytotoxicity in vivo Conversely, in mice chronically infected with CL13, NK cells are immature and residually activated, and following ECTV infection, they do not mature, proliferate, or increase their cytotoxicity. Given the well-established importance of NK cells in resistance to mousepox, these data suggest that the NK cell dysfunction caused by CL13 persistence may contribute to the susceptibility of CL13-infected mice to mousepox. Whether chronic infections similarly affect NK cells in humans should be explored.IMPORTANCE Infection of adult mice with the clone 13 (CL13) strain of lymphocytic choriomeningitis virus (LCMV) is extensively used as a model of chronic infection. In this paper, we show that mice chronically infected with CL13 succumb to challenge with ectromelia virus (ECTV; the agent of mousepox) and that natural killer (NK) cells in CL13-infected mice are reduced in numbers and have an immature and partially activated phenotype but do respond to ECTV. These data may provide additional clues why humans chronically infected with certain pathogens are less resistant to viral diseases.

Loss of Resistance to Mousepox during Chronic Lymphocytic Choriomeningitis Virus Infection Is Associated with Impaired T-Cell Responses and Can Be Rescued by Immunization.

It is well established that chronic viral infections can cause immune suppression, resulting in increased susceptibility to other infectious diseases. However, the effects of chronic viral infection on T-cell responses and vaccination against highly pathogenic viruses are not well understood. We have recently shown that C57BL/6 (B6) mice lose their natural resistance to wild-type (WT) ectromelia virus (ECTV) when chronically infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13). Here we compared the T-cell response to ECTV in previously immunologically naive mice that were chronically infected with CL13 or that were convalescent from acute infection with the Armstrong (Arm) strain of LCMV. Our results show that mice that were chronically infected with CL13 but not those that had recovered from Arm infection have highly defective ECTV-specific CD8+ and CD4+ T-cell responses to WT ECTV. These defects are at least partly due to the chronic infection environment. In contrast to mice infected with WT ECTV, mice chronically infected with CL13 survived without signs of disease when infected with ECTV-Δ036, a mutant ECTV strain that is highly attenuated. Strikingly, mice chronically infected with CL13 mounted a strong CD8+ T-cell response to ECTV-Δ036 and survived without signs of disease after a subsequent challenge with WT ECTV. Our work suggests that enhanced susceptibility to acute viral infections in chronically infected individuals can be partly due to poor T-cell responses but that sufficient T-cell function can be recovered and resistance to acute infection can be restored by immunization with highly attenuated vaccines.IMPORTANCE Chronic viral infections may result in immunosuppression and enhanced susceptibility to infections with other pathogens. For example, we have recently shown that mice chronically infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13) are highly susceptible to mousepox, a disease that is caused by ectromelia virus and that is the mouse homolog of human smallpox. Here we show chronic CL13 infection severely disrupts the expansion, proliferation, activation, and cytotoxicity of T cells in response due at least in part to the suppressive effects of the chronic infection milieu. Notably, despite this profound immunodeficiency, mice chronically infected with CL13 could be protected by vaccination with a highly attenuated variant of ECTV. These results demonstrate that protective vaccination of immunosuppressed individuals is possible, provided that proper immunization tools are used.