Relatively preserved functional immune capacity with standard COVID-19 vaccine regimen in people living with HIV.

Introduction: People living with HIV (PLWH) are at a higher risk of severe disease with SARS-CoV-2 virus infection. COVID-19 vaccines are effective in most PLWH. However, suboptimal immune responses to the standard two-shot regimen are a concern, especially for those with moderate to severe immunodeficiency. An additional dose is recommended as part of the extended primary series in Taiwan. Herein, we study the efficacy of this additional shot in PLWH with mild immunodeficiency compared to that in healthy non-HIV people. Methods: In total, 72 PLWH that were asymptomatic or with mild immunodeficiency (CD4 counts ≥200/mm3) and suppressed virology, and 362 healthcare workers of our hospital were enrolled. None of the participants had a history of SARS-CoV-2 infection. They received mRNA-1273 and ChAdOx1 vaccines. Anti-SARS-CoV-2 neutralizing and anti-Spike IgG antibodies, and SARS-CoV-2-specific T cell responses were evaluated. Results: The standard two-shot regimen elicited lower responses in PLWH than the healthcare workers without HIV infection, although the difference was statistically insignificant. They had comparable levels of neutralizing and anti-Spike antibodies and comparable effector CD4+ and CD8+ T cell responses. The third shot boosted the SARS-CoV-2 immunity significantly more with better antibody responses and higher IFN-γ and IL-2 responses of the CD4+ and CD8+ T cells in PLWH compared to those without HIV. Upon in vitro stimulation with extracted Wuhan strain SARS-CoV-2 proteins, CD8+ T cells from PLWH after 3 shots had more durable effector responses than the non-HIV controls with extended time of stimulation. Conclusion: This subtle difference between PLWH and non-HIV people implied immune exhaustion with two shots in non-HIV people. Slightly compromised immunity in PLWH indeed preserved the functional capacity for further response to the third shot or natural infection.

An IL-17-EGFR-TRAF4 axis contributes to the alleviation of lung inflammation in severe influenza.

Excessive inflammation is a postulated cause of severe disease and death in respiratory virus infections. In response to severe influenza virus infection, adoptively transferred naïve hemagglutinin-specific CD4+ T cells from CD4+ TCR-transgenic 6.5 mice drive an IFN-γ-producing Th1 response in wild-type mice. It helps in virus clearance but also causes collateral damage and disease aggravation. The donor 6.5 mice have all the CD4+ T cells with TCR specificity toward influenza hemagglutinin. Still, the infected 6.5 mice do not suffer from robust inflammation and grave outcome. The initial Th1 response wanes with time, and a prominent Th17 response of recent thymic emigrants alleviates inflammation and bestows protection in 6.5 mice. Our results suggest that viral neuraminidase-activated TGF-ß of the Th1 cells guides the Th17 evolution, and IL-17 signaling through the non-canonical IL-17 receptor EGFR activates the scaffold protein TRAF4 more than TRAF6 during alleviation of lung inflammation in severe influenza.

The origin of regulatory from the effector cells in LAG-3-marked Th1 immunity against severe influenza virus infection.

In severe respiratory virus infections, including influenza, an exaggerated host immune response has been linked to the severe disease and death. Control of the overwhelming immune response is thus essential. Efforts with broad-spectrum immunosuppressive agents such as steroids are disappointing. A better understanding of host immune response using animal experimental system is required to avoid undesired outcome of experimental manipulation. Following severe influenza virus infection in influenza hemagglutinin antigen-specific transgenic mouse experimental model, step-wise evolving cells from a pool of naïve hemagglutinin-specific CD4+ T cells were studied for phenotypic, genomic, and functional characterization in vivo. Naïve CD4+ T cells respond with Th1 commitment in the absolute majority. They first develop into LAG-3Med IFN-γ-secreting Th1 effectors and then evolve into LAG-3High IFN-γ-not-secreting regulators with increasing LAG-3 expression upon continuous activation and cell division. The LAG-3Med IFN-γ-secreting effectors contribute to inflammation, boost inflammatory response of cognate antigen-specific CD8+ T cells, and aggravate the disease despite facilitated virus clearance. In contrast, LAG-3High regulators do not contribute to inflammation, suppress CD8+ T cell inflammatory response, alleviate lung pathology, and ameliorate the disease with preserved virus clearance. Moderated CD8+ T cells retain proliferative capacity, and persist beyond virus clearance. Such moderation is distinct from Foxp-3+ regulator-mediated suppression, which suppresses proliferative and inflammatory responses of the CD8+ T cells and impairs virus clearance with inflammation alleviation. Origin of regulatory from the effector cells of LAG-3-marked Th1 immunity alleviates lung inflammation without impairment of virus eradication.

Role of Pneumococcal NanC in the Severe Disease of Streptococcus pneumoniae Superinfection with Influenza.

Bacterial superinfection aggravates the disease of influenza. Streptococcus pneumoniae is the most common bacterial pathogen. Synergistic virulence has been demonstrated between influenza neuraminidase and pneumococcal NanA and NanB. NanC, the other pneumococcal neuraminidase infrequently present in clinical isolates, is not well characterized. In this study, we report that superinfection with a NanC-negative pneumococcus strain suppresses anti-influenza immunity and impairs viral clearance with higher TGF-ß activation in mice. Bacterial load in the lungs also increases as the host immunity is suppressed. NanC-positive isogenic mutant reverses wild type S. pneumoniae-mediated immune suppression and facilitates virus clearance. However, it causes more severe disease as the augmented inflammation causes collateral damage. Both virus-mediated damage and immune response-mediated inflammation are important for pathogenesis of severe influenza. Inflammation may be more critical than virus-mediated damage in influenza with bacterial superinfection.

Legacy Effect of Antioxidant N-acetylcysteine in Cellular Senescence of Diet-induced Obesity Mice.

BACKGROUND: Cellular senescence is a state of stable growth arrest triggered by mitogenic and metabolic stressors. Ageing and a high-fat diet (HFD) are proven inducers of senescence in various organs, presenting a challenge for ageing populations worldwide. Our previous study demonstrated that ROS scavenger N-acetylcysteine (NAC) can improve insulin resistance (IR) and chronic inflammation in diet-induced obesity mice, an effect better achieved through early intervention. We, herein, investigate whether NAC can improve cellular senescence in a diet-induced obesity mouse model, and whether a legacy effect is presented with early intervention. MATERIALS AND METHODS: For a twelve-month treatment course, all C57B/L6 mice were fed a chow diet (CD), high-fat high-sucrose diet (HFD), CD+NAC1-12 (NAC intervention 1st-12th month), HFD+NAC1-12, and HFD+NAC1-6 (NAC intervention 1st-6th month). Staticalanalysis was used to analyze the different markers of cellular senescence and inflammation. RESULTS: Throughout the study, the HFD group exhibited significantly increased body weight (BW) and body fat, markers of senescence, decreased motor activity (MA) and impaired glucose tolerance. Compared to the HFD group, the HFD+NAC1-12 group exhibited increased MA, decreased BW and body fat, improved glucose tolerance, and decreased senescence markers.The HFD+NAC1-6 group showed similar effects to the HFD+NAC1-12 group, despite discontinuing NAC for 6 months. Our study showed that NAC significantly increased MA in both HFD+NAC1-12 and HFD+NAC1-6 groups, and improved HFD-induced mitochondrial and intracellular ROS expression, DNA and protein oxidative damage, and adipose tissue inflammation. CONCLUSION: Legacy effect was indeed presented in HFD-induced cellular senescence with NAC intervention, with possible mechanisms being persistently increased motor activity and anti-oxidative stress effects.

Abrogation of Toll-Like Receptor 4 Mitigates Obesity-Induced Oxidative Stress, Proinflammation, and Insulin Resistance Through Metabolic Reprogramming of Mitochondria in Adipose Tissue.

Aims: Obesity-induced excessive visceral fat (VF) accumulation is associated with insulin resistance (IR), systemic oxidative stress, and chronic inflammation. As toll-like receptor 4 (TLR4) plays an important role in innate immunity, we herein investigate the effect of TLR4 knockout (T4KO) in a high-fat high-sucrose diet (HFD)-induced obesity mouse model. Results: C57BL6 wild-type (WT) and T4KO mice were fed with either control diet (CD) or HFD for 12 months, rendering four experimental groups: WT+CD, WT+HFD, T4KO+CD, and T4KO+HFD. Compared with WT+CD, WT+HFD demonstrated significant increase in VF accumulation, oxidative damage, M1/M2 macrophage ratio, chronic inflammation, and development of IR. Compared with WT+HFD, T4KO+HFD presented increased BW and body fat with higher subcutaneous fat (SF)/VF ratio, but lower body temperature, as well as decreased oxidative damage, M1/M2 macrophage ratio, chronic inflammation, and IR. Unlike WT+HFD, T4KO+HFD exhibited an increase in mitochondrial electron transport chain activity but a decrease of uncoupling protein 2 (UCP2) level. While T4KO hindered HFD-induced increasing mitochondrial oxygen consumption rate, a shift toward a higher extracellular acidification rate in VF was observed. Notably, T4KO inhibits HFD-induced mitochondrial translocation of nuclear factor of activated T cells 2 (NFATC2), which contributed to mitochondrial metabolic reprogramming. Both fat distribution shifting from VF to SF and mitochondrial metabolic reprogramming may alleviate systemic oxidative stress and chronic inflammation. Innovation and Conclusion: Abrogation of TLR4 contributes to reduction of oxidative stress through metabolic reprogramming of mitochondria in VF, mitigating obesity-induced IR. The study provides critical insight into associating innate immunity-mitochondria interplay with prevention of diabetes.

Effect of aloin on viral neuraminidase and hemagglutinin-specific T cell immunity in acute influenza.

BACKGROUND: Millions of people are infected by the influenza virus worldwide every year. Current selections of anti-influenza agents are limited and their effectiveness and drug resistance are still of concern. PURPOSE: Investigation on in vitro and in vivo effect of aloin from Aloe vera leaves against influenza virus infection. METHODS: In vitro antiviral property of aloin was measured by plaque reduction assay in which MDCK cells were infected with oseltamivir-sensitive A(H1N1)pdm09, oseltamivir-resistant A(H1N1)pdm09, H1N1 or H3N2 influenza A or with influenza B viruses in the presence of aloin. In vivo activity was tested in H1N1 influenza virus infected mice. Aloin-mediated inhibition of influenza neuraminidase activity was tested by MUNANA assay. Aloin treatment-mediated modulation of anti-influenza immunity was tested by the study of hemagglutinin-specific T cells in vivo. RESULTS: Aloin significantly reduced in vitro infection by all the tested strains of influenza viruses, including oseltamivir-resistant A(H1N1)pdm09 influenza viruses, with an average IC50 value 91.83 ± 18.97 µM. In H1N1 influenza virus infected mice, aloin treatment (intraperitoneal, once daily for 5 days) reduced virus load in the lungs and attenuated body weight loss and mortality. Adjuvant aloin treatment also improved the outcome with delayed oseltamivir treatment. Aloin inhibited viral neuraminidase and impeded neuraminidase-mediated TGF-ß activation. Viral neuraminidase mediated immune suppression with TGF-ß was constrained and influenza hemagglutinin-specific T cell immunity was increased. There was more infiltration of hemagglutinin-specific CD4+ and CD8+ T cells in the lungs and their production of effector cytokines IFN-γ and TNF-α was boosted. CONCLUSION: Aloin from Aloe vera leaves is a potent anti-influenza compound that inhibits viral neuraminidase activity, even of the oseltamivir-resistant influenza virus. With suppression of this virus machinery, aloin boosts host immunity with augmented hemagglutinin-specific T cell response to the infection. In addition, in the context of compromised benefit with delayed oseltamivir treatment, adjuvant aloin treatment ameliorates the disease and improves survival. Taken together, aloin has the potential to be further evaluated for clinical applications in human influenza.

Early intervention of N-acetylcysteine better improves insulin resistance in diet-induced obesity mice.

Reactive oxygen species (ROS) plays a crucial role in pathogenesis of insulin resistance (IR) and type 2 diabetes. In the United Kingdom, Prospective Diabetes Study and its 10-year post-trial monitoring, a beneficial effect of early optimisation of blood glucose control is clearly demonstrated. In this study, we investigated whether ROS scavenger N-acetylcysteine (NAC) and the time point of intervention can affect IR in a diet-induced obesity mouse model. Male C57B/L6 mice were fed chow diet (CD), high-fat high-sucrose diet (HFD), CD + NAC1-6 (NAC intervention 1st to 6th month), HFD + NAC1-6, and HFD + NAC3-6 (NAC intervention 3rd to 6th month) for a 6-month treatment course. HFD group showed significantly increased body weight (BW) and body fat, decreased motor activity (MA), impaired intraperitoneal glucose tolerance test (IPGTT), and insulin tolerance test (IPITT) throughout the study. HFD + NAC1-6, as compared with HFD group, had increased MA, improved IPGTT and IPITT since first month, followed by decreased BW and body fat. HFD + NAC3-6 group, although showed improved IPGTT and IPITT than HFD group, still had higher BW, decreased MA, and impaired IPGTT and IPITT as compared with HFD + NAC1-6 at the end of the study. NAC significantly increased MA, and ameliorated the HFD-induced mitochondrial and intracellular ROS expression, DNA and protein oxidative damage, and adipose tissue inflammation. We concluded that ROS scavenger can improve IR and chronic inflammation in diet-induced obesity mice. This action is likely better expressed through early intervention. The mechanism is probably through a virtuous circle of suppressed oxidative stress, and increased motor activity, which helps to reduce body fat.

Rapamycin adjuvant and exacerbation of severe influenza in an experimental mouse model.

Influenza virus infection often causes severe disease and acute respiratory distress syndrome. It is a common belief that overwhelming immune response contributes to the severe illness. Physicians and researchers have put forth immune modulation as salvage therapy for better recovery. However, empiric corticosteroid failed in both humans and animal models. Reported success with Rapamycin in humans prompted a comprehensive animal study and mechanistic dissection. Here we report the effect of Rapamycin alone or in combination with Oseltamivir for severe influenza in BALB/c mice. We found that Rapamycin had no antiviral effect against H1N1, H3N2 and novel-H1N1 influenza viruses in vitro. Rapamycin alone aggravated the severe disease of PR8 H1N1 influenza virus infection in mice. Timely Oseltamivir anti-viral therapy abolished the disease. Delayed Oseltamivir treatment could not prevent severe illness and Rapamycin adjuvant was associated with exacerbated disease. Rapamycin adjuvant suppressed influenza hemagglutinin antigen-specific T cell immunity and impaired virus clearance from the lungs. It also resulted in intensified lung pathology with increased intra-alveolar edema and hyaline deposition. Rapamycin may work as the salvage therapy for severe influenza but it is very difficult to define the appropriate window for such treatment to take effect.

Sterilizing immunity to influenza virus infection requires local antigen-specific T cell response in the lungs.

Sterilizing immunity is a unique immune status, which prevents effective virus infection into the host. It is different from the immunity that allows infection but with subsequent successful eradication of the virus. Pre-infection induces sterilizing immunity to homologous influenza virus challenge in ferret. In our antigen-specific experimental system, mice pre-infected with PR8 influenza virus through nasal route are likewise resistant to reinfection of the same strain of virus. The virus is cleared before establishment of effective infection. Intramuscular influenza virus injection confers protection against re-infection with facilitated virus clearance but not sterilizing immunity. Pre-infection and intramuscular injection generates comparable innate immunity and antibody response, but only pre-infection induces virus receptor reduction and efficient antigen-specific T cell response in the lungs. Pre-infection with nH1N1 influenza virus induces virus receptor reduction but not PR8-specific T cell immune response in the lungs and cannot prevent infection of PR8 influenza virus. Pre-infection with PR8 virus induced PR8-specific T cell response in the lungs but cannot prevent infection of nH1N1 virus either. These results reveal that antigen-specific T cell immunity is required for sterilizing immunity.

High-fat diet induces toll-like receptor 4-dependent macrophage/microglial cell activation and retinal impairment.

PURPOSE: The toll-like receptor 4 (TLR4) signaling pathway is involved in chronic inflammation and insulin resistance, which are associated with obesity and diabetes mellitus. In the present study, a model of high-fat diet (HFD) feeding of mice was used to investigate the role of TLR4 in overnutrition- and obesity-associated inflammation and infiltration of macrophages and microglia in the retina. METHODS: Wild-type C57BL/6 and TLR4 knockout (TLR4KO; B6.B10ScN-Tlr4(lps-del)/JthJ) mice were fed a HFD or control chow diet (CD) for 6 months. The TLR4 expression, the relative increase in macrophages/microglia (CD11b(+) and CD45(+) cells), the presence of markers of oxidative stress (gp91phox and malondialdehyde; MDA), and DNA damage (phosphorylated histone H2AX; γH2AX) were assessed by real-time PCR and immunofluorescence studies. RESULTS: The HFD for 6 months showed increased obesity, glucose intolerance, and insulin resistance in mice. Toll-like receptor 4 expression was found in vascular pericytes at the inner retina. Increased CD11b(+) and CD45(+) cells, phosphorylated NF-κB, interleukin-6, gp91phox, MDA, and γH2AX were observed in the retina of mice fed a HFD compared to CD counterparts. TLR4KO mice did not show the adverse effects of HFD. CONCLUSIONS: Our results indicate that HFD-induced macrophage/microglial cell activation and retinal impairment were reduced in the absence of TLR4. The findings suggest that TLR4 is implicated in the pathogenesis of retinal diseases caused by metabolic disorders.

IL-10 inhibits neuraminidase-activated TGF-ß and facilitates Th1 phenotype during early phase of infection.

Th1 cells control their activity by producing regulatory IL-10. Here we report that Th1 cell-derived IL-10 facilitates their expansion and, in addition, augments Th1 cell production of IFN-γ, TNF-α and IL-2 during the early phase of influenza. In our antigen-specific mouse experimental system, influenza haemagglutinin-specific CD4(+) T cells respond to infection with the induction of T-bet, and produce both IFN-γ and IL-10. In the early phase of infection, an abundance of viral neuraminidase causes TGF-ß activation of haemagglutinin-specific CD4(+) T cells. CD4(+) T-cell-derived IL-10 inhibits neuraminidase-driven TGF-ß activation and counteracts the virus-mediated immune suppression. As the host eradicates the virus, neuraminidase activity wanes and IL-10 receptors are upregulated on CD4(+) T cells in the late phase of infection. IL-10 then suppresses immune activation and aids in recovery from infection and inflammation. These results reveal a previously unrecognized function of Th1 cell-derived IL-10 in vivo.

Altered T-bet dominance in IFN-γ-decoupled CD4+ T cells with attenuated cytokine storm and preserved memory in influenza.

Cytokine storm has been postulated as one of the major causes of mortality in patients with severe respiratory viral infections such as influenza. With the help of an influenza Ag- specific mouse experimental system, we report that CD4(+) T cells contribute effector cytokines leading to lung inflammation in acute influenza. Although virus can no longer be detected from tissues 14 d postinfection, virus-derived Ag continues to drive a CD4(+) T cell response after viral clearance. Ag-specific CD4(+) T cells proliferate and evolve into memory CD4(+) T cells efficiently, but the production of effector cytokines is seriously hampered during this phase. This decoupling of proliferation and effector cytokine production doesn't appear in conjunction with increased suppression by regulatory T cells or decreased induction of transcription factors. Rather, GATA-3 and ROR-γt levels are elevated when compared with cells that have effector cytokine production. T-bet dominance over GATA-3 and ROR-γt decreases with the disarmament of effector cytokine production. Importantly, upon reinfection, these decoupled cells produce elevated levels of IFN-γ and were effective in virus eradication. These results provide a mechanism through altered T-bet dominance to dampen the cytokine storm without impeding the generation of memory T cells in influenza virus infection.