Enhanced SARS-CoV-2-Specific CD4+ T Cell Activation and Multifunctionality in Late Convalescent COVID-19 Individuals

Background: Examination of CD4(+) T cell responses during the natural course of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection offers useful information for the improvement of vaccination strategies against this virus and the protective effect of these T cells. Methods: We characterized the SARS-CoV-2-specific CD4(+) T cell activation marker, multifunctional cytokine and cytotoxic marker expression in recovered coronavirus disease 2019 (COVID-19) individuals. Results: CD4(+) T-cell responses in late convalescent (>6 months of diagnosis) individuals are characterized by elevated frequencies of activated as well as mono, dual- and multi-functional Th1 and Th17 CD4(+) T cells in comparison to early convalescent (<1 month of diagnosis) individuals following stimulation with SARS-CoV-2-specific antigens. Similarly, the frequencies of cytotoxic marker expressing CD4(+) T cells were also enhanced in late convalescent compared to early convalescent individuals. Conclusion: Our findings from a low-to middle-income country suggest protective adaptive immune responses following natural infection of SARS-CoV-2 are elevated even at six months following initial symptoms, indicating the CD4(+) T cell mediated immune protection lasts for six months or more in natural infection

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

Characterization of memory T cell subsets and common γ−chain cytokines in convalescent COVID-19 individuals

T cells are thought to be an important correlates of protection against SARS‐CoV2 infection. However, the composition of T cell subsets in convalescent individuals of SARS‐CoV2 infection has not been well studied. The authors determined the lymphocyte absolute counts, the frequency of memory T cell subsets, and the plasma levels of common γ−chain in 7 groups of COVID‐19 individuals, based on days since RT‐PCR confirmation of SARS‐CoV‐2 infection. The data show that both absolute counts and frequencies of lymphocytes as well as, the frequencies of CD4(+) central and effector memory cells increased, and the frequencies of CD4(+) naïve T cells, transitional memory, stem cell memory T cells, and regulatory cells decreased from Days 15–30 to Days 61–90 and plateaued thereafter. In addition, the frequencies of CD8(+) central memory, effector, and terminal effector memory T cells increased, and the frequencies of CD8(+) naïve cells, transitional memory, and stem cell memory T cells decreased from Days 15–30 to Days 61–90 and plateaued thereafter. The plasma levels of IL‐2, IL‐7, IL‐15, and IL‐21—common γc cytokines started decreasing from Days 15–30 till Days 151–180. Severe COVID‐19 patients exhibit decreased levels of lymphocyte counts and frequencies, higher frequencies of naïve cells, regulatory T cells, lower frequencies of central memory, effector memory, and stem cell memory, and elevated plasma levels of IL‐2, IL‐7, IL‐15, and IL‐21. Finally, there was a significant correlation between memory T cell subsets and common γc cytokines. Thus, the study provides evidence of alterations in lymphocyte counts, memory T cell subset frequencies, and common γ−chain cytokines in convalescent COVID‐19 individuals

An adverse outcome pathway for immune-mediated and allergic hepatitis: a case study with the NSAID diclofenac

Many drugs have the potential to cause drug-induced liver injury (DILI); however, underlying mechanisms are diverse. The concept of adverse outcome pathways (AOPs) has become instrumental for risk assessment of drug class effects. We report AOPs specific for immune-mediated and drug hypersensitivity/allergic hepatitis by considering genomic, histo- and clinical pathology data of mice and dogs treated with diclofenac. The findings are relevant for other NSAIDs and drugs undergoing iminoquinone and quinone reactive metabolite formation. We define reactive metabolites catalyzed by CYP monooxygenase and myeloperoxidases of neutrophils and Kupffer cells as well as acyl glucuronides produced by uridine diphosphoglucuronosyl transferase as molecular initiating events (MIE). The reactive metabolites bind to proteins and act as neo-antigen and involve antigen-presenting cells to elicit B- and T-cell responses. Given the diverse immune systems between mice and dogs, six different key events (KEs) at the cellular and up to four KEs at the organ level are defined with mechanistic plausibility for the onset and progression of liver inflammation. With mice, cellular stress response, interferon gamma-, adipocytokine- and chemokine signaling provided a rationale for the AOP of immune-mediated hepatitis. With dogs, an erroneous programming of the innate and adaptive immune response resulted in mast cell activation; their infiltration into liver parenchyma and the shift to M2-polarized Kupffer cells signify allergic hepatitis and the occurrence of granulomas of the liver. Taken together, diclofenac induces divergent immune responses among two important preclinical animal species, and the injury pattern seen among clinical cases confirms the relevance of the developed AOP for immune-mediated hepatitis

Whole Genome Transcript Profiling of Drug Induced Steatosis in Rats Reveals a Gene Signature Predictive of Outcome

<div><p>Drug induced steatosis (DIS) is characterised by excess triglyceride accumulation in the form of lipid droplets (LD) in liver cells. To explore mechanisms underlying DIS we interrogated the publically available microarray data from the Japanese Toxicogenomics Project (TGP) to study comprehensively whole genome gene expression changes in the liver of treated rats. For this purpose a total of 17 and 12 drugs which are diverse in molecular structure and mode of action were considered based on their ability to cause either steatosis or phospholipidosis, respectively, while 7 drugs served as negative controls. In our efforts we focused on 200 genes which are considered to be mechanistically relevant in the process of lipid droplet biogenesis in hepatocytes as recently published (Sahini and Borlak, 2014). Based on mechanistic considerations we identified 19 genes which displayed dose dependent responses while 10 genes showed time dependency. Importantly, the present study defined 9 genes (ANGPTL4, FABP7, FADS1, FGF21, GOT1, LDLR, GK, STAT3, and PKLR) as signature genes to predict DIS. Moreover, cross tabulation revealed 9 genes to be regulated ≥10 times amongst the various conditions and included genes linked to glucose metabolism, lipid transport and lipogenesis as well as signalling events. Additionally, a comparison between drugs causing phospholipidosis and/or steatosis revealed 26 genes to be regulated in common including 4 signature genes to predict DIS (PKLR, GK, FABP7 and FADS1). Furthermore, a comparison between <i>in vivo</i> single dose (3, 6, 9 and 24 h) and findings from rat hepatocyte studies (2 h, 8 h, 24 h) identified 10 genes which are regulated in common and contained 2 DIS signature genes (FABP7, FGF21). Altogether, our studies provide comprehensive information on mechanistically linked gene expression changes of a range of drugs causing steatosis and phospholipidosis and encourage the screening of DIS signature genes at the preclinical stage.</p></div

Diclofenac Disrupts the Circadian Clock and through Complex Cross-Talks Aggravates Immune-Mediated Liver Injury—A Repeated Dose Study in Minipigs for 28 Days

Diclofenac effectively reduces pain and inflammation; however, its use is associated with hepato- and nephrotoxicity. To delineate mechanisms of injury, we investigated a clinically relevant (3 mg/kg) and high-dose (15 mg/kg) in minipigs for 4 weeks. Initially, serum biochemistries and blood-smears indicated an inflammatory response but returned to normal after 4 weeks of treatment. Notwithstanding, histopathology revealed drug-induced hepatitis, marked glycogen depletion, necrosis and steatosis. Strikingly, the genomic study revealed diclofenac to desynchronize the liver clock with manifest inductions of its components CLOCK, NPAS2 and BMAL1. The > 4-fold induced CRY1 expression underscored an activated core-loop, and the dose dependent > 60% reduction in PER2mRNA repressed the negative feedback loop; however, it exacerbated hepatotoxicity. Bioinformatics enabled the construction of gene-regulatory networks, and we linked the disruption of the liver-clock to impaired glycogenesis, lipid metabolism and the control of immune responses, as shown by the 3-, 6- and 8-fold induced expression of pro-inflammatory CXCL2, lysozyme and ß-defensin. Additionally, diclofenac treatment caused adrenocortical hypertrophy and thymic atrophy, and we evidenced induced glucocorticoid receptor (GR) activity by immunohistochemistry. Given that REV-ERB connects the circadian clock with hepatic GR, its > 80% repression alleviated immune responses as manifested by repressed expressions of CXCL9(90%), CCL8(60%) and RSAD2(70%). Together, we propose a circuitry, whereby diclofenac desynchronizes the liver clock in the control of the hepatic metabolism and immune response

Venn diagram of DEGs associated with steatosis and phospholipidosis.

<p>Panel A: DEGs associated with phospholipidosis under all conditions (single dose at 3, 6, 9 and 24 h and repeated treatment (3, 7, 14 and 28 days) at the low, mid and high dose for 12 phospholipidosis drugs). Note, none of the DEGs were commonly regulated amongst phospholipidosis drugs. Panel B: DEGs common amongst phospholipidosis and steatosis drugs. By applying less stringent criteria (regulation in any of the conditions, either by dose or time) a total of 26 genes were commonly regulated. Panel C: PPI network among the 26 common genes. Of these 58% proteins interact with each other. The strength of association is depicted with the thickness of the blue line between the interacting proteins (STRING 9.1, confidence view).</p