Exhausted and Apoptotic BALF T Cells in Proinflammatory Airway Milieu at Acute Phase of Severe Mycoplasma Pneumoniae Pneumonia in Children

Severe mycoplasma pneumoniae pneumonia (MPP) in children presents with serious clinical complications. Without proper and prompt intervention, it could lead to deadly consequences. Dynamics of the inflammatory airway milieu and activation status of immune cells were believed to be the hallmark of the pathogenesis and progress of the disease. In this study, by employing the T-cell sorting and mRNA microarray, we were able to define the main feature of the chemokine/cytokine expression and the unique characteristics of T cells in the bronchoalveolar lavage fluid (BALF) from severe MPP patients at acute phase. Our study for the first time delineated the molecular changes in isolated BALF T cells in severe MPP children with respect to the cytokine/chemokine expression, cell activation, exhaustion, and apoptosis. By comparing the BALF aqueous expression of cytokines/chemokines with that in sorted T cells, our data give a preliminary clue capable of finishing out the possible cell source of the proinflammatory cytokines/chemokines from the BALF mixture. Meanwhile, our data provide a distinctively pellucid expression profile particularly belonging to the isolated BALF T cells demonstrating that in the inflammatory airway, overactivated T cells were exhausted and on the verge of apoptotic progress.

SARS-CoV-2 diverges from other betacoronaviruses in only partially activating the IRE1α/XBP1 ER stress pathway in human lung-derived cells

Despite the efficacy of vaccines, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed over 5 million individuals worldwide and continues to spread in countries where the vaccines are not yet widely available or its citizens are hesitant to become vaccinated. Therefore, it is critical to unravel the molecular mechanisms that allow SARS-CoV-2 and other coronaviruses to infect and overtake the host machinery of human cells. Coronavirus replication triggers endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR), a key host cell pathway widely believed essential for viral replication. We examined the activation status and requirement of the master UPR sensor IRE1α kinase/RNase and its downstream transcription factor effector XBP1s, which is processed through an IRE1α-mediated mRNA splicing event, in human lung-derived cells infected with betacoronaviruses. We found human respiratory coronavirus OC43 (HCoV-OC43), Middle East respiratory syndrome coronavirus (MERS-CoV), and the murine coronavirus (MHV) all induce ER stress and strongly trigger the kinase and RNase activities of IRE1α as well as XBP1 splicing. In contrast, SARS-CoV-2 only partially activates IRE1α whereby it autophosphorylates, but its RNase fails to splice XBP1. Moreover, IRE1α was dispensable for optimal replication in human cells for all coronaviruses tested. Our findings demonstrate that IRE1α activation status differs upon infection with distinct betacoronaviruses and is not essential for efficient replication of any of them. Our data suggest that SARS-CoV-2 actively inhibits the RNase of autophosphorylated IRE1α through an unknown mechanism, perhaps as a strategy to eliminate detection by the host immune system.

Moderate to intense physical activity is associated with improved clinical, CD4/CD8 ratio and immune activation status in HIV infected patients on ART

Background Physical activity has anti-inflammatory effects and reduces morbidity and mortality in general population, but its role in the clinical, CD4/CD8 ratio and immune activation status in HIV-infected patients has been poorly studied. Methods A cross-sectional study was carried out in a cohort of 155 HIV-infected patients on stable ART to compare clinical, biochemical, CD4/CD8 ratio and immune-activation status according to their physical activity in the last two years (sedentary/low vs. moderate/intense) assessed by the iPAQ. A binary logistic regression and mixed ANOVA were performed to evaluate the impact of levels of physical activity on CD4/CD8 ratio. Results In our series 77 (49.7%) out of 155 patients were sedentary and 78 (50.3%) practiced moderate/intense physical activity. Moderate/intense physical activity was associated with lower CDC HIV-stage (p=0.046), better metabolic control (lower BMI, p=0.024; glucose, p=0.024; and triglyceride, p=0.002), higher CD3 +CD4 + T lymphocytes (p=0.016), lower CD8 + T lymphocytes (p=0.018), higher CD4/CD8 ratio (p=0.001), lower CD4 +CD8 + (p=0.026), CD4 +CD86 + (p=0.045), CD4 +HLA-DR + (p=0.011), CD8 +HLA-DR + (p=0.048) T lymphocytes and CD16 +HLA-DR + NK cells (p=0.026). Sedentary lifestyle (OR=2.12, p=0.042), CD4 nadir (OR=1.005, p<0.001) and CD8 +CD38 + T cells (OR=1.27, p=0.006) were independently associated with low CD4/CD8 ratio (<0.8). Earlier and more intense CD4/CD8 ratio recovery was observed in patients with higher physical activity in the two-year follow-up with a significant interaction between these variables: F(2, 124) = 3.31, p=0.049, partial η2=0.042). Conclusions Moderate to high physical activity is associated with beneficial health effects, improving metabolic profile and reducing chronic inflammation in patients living with HIV. Although more studies and clinical trials are needed to confirm these findings, healthy lifestyle, including at least moderate physical activity, should be recommended to HIV patients on stable ART.

Impact of Immunonutrition on T Cell Activation: A Randomized Control Study in Cardiac Surgery Patients

Background. Cardiac surgery provokes an intense inflammatory response that can cause an immunosuppressive state and adverse postoperative outcomes. We recently showed that postoperative immunonutrition with glutamine in “fragile” low-risk cardiac surgery patients was associated with a significantly increased level of CD3+ and CD4+ T cells. In order to clarify the biological relevance and clinical importance of these findings, we investigated whether an increase in the CD4+ T cell level was caused by changes in the systemic inflammatory response (caused by surgery or infection) and if it was associated with their activation status.Methods. A randomized control study of low operative risk but “fragile” cardiac surgery patients was performed. Patients were randomized into immunonutrition (IN) and control groups (C). The IN group received normal daily meals plus special immune nutrients for 5 days postoperatively, while the C group received only normal daily meals. Laboratory parameters were investigated before surgery and on the sixth postoperative day and the groups were compared accordingly. The expression of the CD69+ marker was investigated to determine T cell activation status. Serum concentrations of cytokines (interleukin-10 (IL-10), tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6)) and C-reactive protein (CRP) were determined to assess the systemic inflammatory response, while procalcitonin (PCT) levels were evaluated to confirm or deny possible bacterial infection.Results. Fifty-five patients were enrolled in the study. Twenty-seven (49.1%) were randomized in the IN group. Results show that on the sixth postoperative day, the CD4+CD69+ and CD8+CD69+ counts did not differ between the IN and C groups, accordingly 0.25 [0.16–0.50] vs 0.22 [0.13-0.41], p=0.578 and 0.13 [0.06–0.3] vs 0.09 [0.05–0.14], p=0.178. Also, statistically significant differences were not observed in the cytokine levels (IN and C groups: TNF-α 8.13 [7.32–10.31] vs 8.78 [7.65–11.2], p=0.300; IL-6 14.65 [9.28–18.95] vs 12.25 [8.55–22.50], p=0.786; IL-10 5.0 [5.0–5.0] vs 5.0 [5.0–5.0], p=0.343 respectively), which imply that an elevated T cell count is not associated with the systemic inflammatory response. Also, PCT (IN and C groups: 0.03 [0.01–0.09] vs 0.05 [0.03–0.08], p=0.352) and CRP (IN and C groups 62.7 [34.2–106.0] vs 63.7 [32.9–91.0], p=0.840) levels did not differ between the two groups. Moreover, low levels of PCT indicated that the increase in T cell count was not determined by bacterial infection.Conclusions. Our findings showed that CD4+ T cell levels were associated with neither the systemic inflammatory response nor bacterial infection. Secondly, increases in T cells are not accompanied by their activation status. These results suggest a hypothesis that a higher postoperative T cell concentration may be associated with postoperative immunonutrition in low-risk cardiac surgery patients with intact cellular vitality, i.e. “fragile”. However, immunonutrition alone did not affect T cell activation status.

Age Related Differences in Monocyte Subsets and Cytokine Pattern during Acute COVID-19—A Prospective Observational Longitudinal Study

The COVID-19 pandemic drastically highlighted the vulnerability of the elderly population towards viral and other infectious threats, illustrating that aging is accompanied by dysregulated immune responses currently summarized in terms like inflammaging and immunoparalysis. To gain a better understanding on the underlying mechanisms of the age-associated risk of adverse outcome in individuals experiencing a SARS-CoV-2 infection, we analyzed the impact of age on circulating monocyte phenotypes, activation markers and inflammatory cytokines including interleukin 6 (IL-6), IL-8 and tumor necrosis factor (TNF) in the context of COVID-19 disease progression and outcome in 110 patients. Our data indicate no age-associated differences in peripheral monocyte counts or subset composition. However, age and outcome are associated with differences in monocyte activation status. Moreover, a distinct cytokine pattern of IL-6, IL-8 and TNF in elderly survivors versus non-survivors, which consolidates over the time of hospitalization, suggests that older patients with adverse outcomes experience an inappropriate immune response, reminiscent of an inflammaging driven immunoparalysis. Our study underscores the value, necessity and importance of longitudinal monitoring in elderly COVID-19 patients, as dynamic changes after symptom onset can be observed, which allow for a differentiated insight into confounding factors that impact the complex pathogenesis following an infection with SARS-CoV-2.

Cell-Extrinsic Priming Increases Permissiveness of CD4+ T Cells to Human Immunodeficiency Virus Infection by Increasing C–C Chemokine Receptor Type 5 Co-receptor Expression and Cellular Activation Status

The chemokine receptor CCR5 is expressed on multiple cell types, including macrophages, dendritic cells, and T cells, and is the major co-receptor used during HIV transmission. Using a standard αCD3/CD28 in vitro stimulation protocol to render CD4+ T cells from PBMCs permissive to HIV infection, we discovered that the percentage of CCR5+ T cells was significantly elevated in CD4+ T cells when stimulated in the presence of peripheral blood mononuclear cells (PBMCs) as compared to when stimulated as purified CD4+ T cells. This indicated that environmental factors unique to the T-PBMCs condition affect surface expression of CCR5 on CD4+ T cells. Conditioned media from αCD3/CD28-stimulated PBMCs induced CCR5 expression in cultures of unstimulated cells. Cytokine profile analysis of these media suggests IL-12 as an inducer of CCR5 expression. Mass cytometric analysis showed that stimulated T-PBMCs exhibited a uniquely activated phenotype compared to T-Pure. In line with increased CCR5 expression and activation status in stimulated T-PBMCs, CD4+ T cells from these cultures were more susceptible to infection by CCR5-tropic HIV-1 as compared with T-Pure cells. These results suggest that in order to increase ex vivo infection rates of blood-derived CD4+ T cells, standard stimulation protocols used in HIV infection studies should implement T-PBMCs or purified CD4+ T cells should be supplemented with IL-12.

Dynamic alterations in monocyte numbers, subset frequencies and activation markers in acute and convalescent COVID-19 individuals

Monocytes are thought to play an important role in host defence and pathogenesis of COVID-19. However, a comprehensive examination of monocyte numbers and function has not been performed longitudinally in acute and convalescent COVID-19. We examined the absolute counts of monocytes, the frequency of monocyte subsets, the plasma levels of monocyte activation markers using flowcytometry and ELISA in seven groups of COVID-19 individuals, classified based on days since RT-PCR confirmation of SARS-CoV2 infection. Our data shows that the absolute counts of total monocytes and the frequencies of intermediate and non-classical monocytes increases from Days 15–30 to Days 61–90 and plateau thereafter. In contrast, the frequency of classical monocytes decreases from Days 15–30 till Days 121–150. The plasma levels of sCD14, CRP, sCD163 and sTissue Factor (sTF)—all decrease from Days 15–30 till Days 151–180. COVID-19 patients with severe disease exhibit higher levels of monocyte counts and higher frequencies of classical monocytes and lower frequencies of intermediate and non-classical monocytes and elevated plasma levels of sCD14, CRP, sCD163 and sTF in comparison with mild disease. Thus, our study provides evidence of dynamic alterations in monocyte counts, subset frequencies and activation status in acute and convalescent COVID-19 individuals.

PSXII-15 Evaluation of later timing of fixed-time artificial insemination for beef cows when using sex-sorted semen following the 7 & 7 Synch protocol

An experiment was designed to evaluate later timing of fixed-time artificial insemination (FTAI) with sex-sorted semen among postpartum beef cows following the 7 & 7 Synch protocol, with the hypothesis that later timing would result in increased pregnancy rates (P/AI) among cows that expressed estrus prior to FTAI. Beef cows (n = 414) were blocked based on age and days postpartum (DPP) and randomly assigned to receive FTAI at 66 or 72 h after administration of prostaglandin F2α (PG). Estrus was synchronized using the 7 & 7 Synch protocol, which consists of administration of PG (500 μg cloprostenol) and insertion of an intravaginal progesterone-releasing insert (CIDR; 1.38 g progesterone) on Day 0, gonadotropin-releasing hormone (GnRH; 100 μg gonadorelin) on Day 7, and PG coincident with CIDR removal on Day 14. Estrus detection aids (EstrotectTM) were applied to all cows on Day 14, and activation status was recorded at fixed-time artificial insemination (FTAI) on Day 17. All cows that expressed estrus prior to FTAI received sex-sorted semen (4 × 106 cells per unit; SexedULTRA 4MTM). The proportion of cows expressing estrus prior to FTAI did not differ between treatments at this power of test [66 h: 71% (146/205); 72 h: 76% (158/209)]. Additionally, P/AI of estrous cows inseminated with sex-sorted semen did not differ between treatments [66 h: 44% (90/205); 72 h: 39% (82/209)]. In conclusion, later timing of FTAI following the 7 & 7 Synch protocol failed to improve P/AI of estrous cows inseminated with sex-sorted semen.