Corrigendum: Specific TLR-mediated HSP70 activation plays a potential role in host defense against the intestinal parasite Giardia duodenalis.

[This corrects the article DOI: 10.3389/fmicb.2023.1120048.].

Predictive value of peripheral lymphocyte subsets for the disease progression in patients with sepsis.

OBJECTIVE: To investigate the predictive value of peripheral lymphocyte subsets for sepsis progression. METHODS: Patients with sepsis were divided into the improved group (n = 46) and severe group (n = 39) according to disease progression. Flow cytometric analysis was performed to enumerate absolute counts of peripheral lymphocyte subsets. Logistic regression analyses were conducted to identify clinical factors linked to sepsis progression. RESULTS: The absolute counts of peripheral lymphocyte subsets were markedly decreased in septic patients compared with healthy controls. After treatment, the absolute counts of lymphocytes, CD3+ T cells, and CD8+ T cells were restored in the improved group, and reduced in the severe group. Logistic regression analysis indicated that a low CD8+ T cells count was a risk factor for sepsis progression. Receiver operating characteristic curve analysis revealed that CD8+ T cells count had the greatest ability to predict sepsis progression. CONCLUSIONS: The absolute counts of CD3+ T cells, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells were significantly higher in the improved group than the severe group. CD8+ T cells count was predictive of sepsis progression. Lymphopenia and CD8+ T cells depletion were associated with the clinical outcomes of sepsis, suggesting that CD8+ T cells have potential as a predictive biomarker and therapeutic target for patients with sepsis.

Specific TLR-mediated HSP70 activation plays a potential role in host defense against the intestinal parasite Giardia duodenalis.

Giardia duodenalis, an important flagellated noninvasive protozoan parasite, infects the upper small intestine and causes a disease termed giardiasis globally. Few members of the heat shock protein (HSP) family have been shown to function as potential defenders against microbial pathogens, while such information is lacking for Giardia. Here we initially screened and indicated that in vitro Giardia challenge induced a marked early upregulation of HSP70 in intestinal epithelial cells (IECs). As noted previously, apoptotic resistance, nitric oxide (NO)-dependent cytostatic effect and parasite clearance, and epithelial barrier integrity represent effective anti-Giardia host defense mechanisms. We then explored the function of HSP70 in modulating apoptosis, NO release, and tight junction (TJ) protein levels in Giardia-IEC interactions. HSP70 inhibition by quercetin promoted Giardia-induced IEC apoptosis, viability decrease, NO release reduction, and ZO-1 and occludin downregulation, while the agonist celastrol could reverse these Giardia-evoked effects. The results demonstrated that HSP70 played a previously unrecognized and important role in regulating anti-Giardia host defense via attenuating apoptosis, promoting cell survival, and maintaining NO and TJ levels. Owing to the significance of apoptotic resistance among those defense-related factors mentioned earlier, we then elucidated the anti-apoptotic mechanism of HSP70. It was evident that HSP70 could negatively regulate apoptosis in an intrinsic way via direct inhibition of Apaf-1 or ROS-Bax/Bcl-2-Apaf-1 axis, and in an extrinsic way via cIAP2-mediated inhibition of RIP1 activity. Most importantly, it was confirmed that HSP70 exerted its host defense function by downregulating apoptosis via Toll-like receptor 4 (TLR4) activation, upregulating NO release via TLR4/TLR2 activation, and upregulating TJ protein expression via TLR2 activation. HSP70 represented a checkpoint regulator providing the crucial link between specific TLR activation and anti-Giardia host defense responses. Strikingly, independent of the checkpoint role of HSP70, TLR4 activation was proven to downregulate TJ protein expression, and TLR2 activation to accelerate apoptosis. Altogether, this study identified HSP70 as a potentially vital defender against Giardia, and revealed its correlation with specific TLR activation. The clinical importance of HSP70 has been extensively demonstrated, while its role as an effective therapeutic target in human giardiasis remains elusive and thus needs to be further clarified.

ROS-AMPK/mTOR-dependent enterocyte autophagy is involved in the regulation of Giardia infection-related tight junction protein and nitric oxide levels.

Giardia duodenalis, a cosmopolitan noninvasive protozoan parasite of zoonotic concern and public health importance, infects the upper portions of the small intestine and causes one of the most common gastrointestinal diseases globally termed giardiasis, especially in situations lacking safe drinking water and adequate sanitation services. The pathogenesis of giardiasis is complex and involves multiple factors from the interaction of Giardia and intestinal epithelial cells (IECs). Autophagy is an evolutionarily conserved catabolic pathway that involves multiple pathological conditions including infection. Thus far, it remains uncertain if autophagy occurs in Giardia-infected IECs and if autophagic process is associated with the pathogenic factors of giardiasis, such as tight junction (TJ) barrier defects and nitric oxide (NO) release of IECs. Here Giardia-in vitro exposed IECs showed upregulation of a series of autophagy-related molecules, such as LC3, Beclin1, Atg7, Atg16L1, and ULK1, and downregulation of p62 protein. IEC autophagy induced by Giardia was further assessed by using autophagy flux inhibitor, chloroquine (CQ), with the ratio of LC3-II/LC3-I significantly increased and downregulated p62 significantly reversed. Inhibition of autophagy by 3-methyladenine (3-MA) rather than CQ could markedly reverse Giardia-induced downregulation of TJ proteins (claudin-1, claudin-4, occludin, and ZO-1; also known as epithelial cell markers) and NO release, implying the involvement of early-stage autophagy in TJ/NO regulation. We subsequently confirmed the role of ROS-mediated AMPK/mTOR signaling in modulating Giardia-induced autophagy, TJ protein expression, and NO release. In turn, impairment of early-stage autophagy by 3-MA and late-stage autophagy by CQ both exhibited an exacerbated effect on ROS accumulation in IECs. Collectively, we present the first attempt to link the occurrence of IEC autophagy with Giardia infection in vitro, and provides novel insights into the contribution of ROS-AMPK/mTOR-dependent autophagy to Giardia infection-related downregulation of TJ protein and NO levels.

Giardia duodenalis and Its Secreted PPIB Trigger Inflammasome Activation and Pyroptosis in Macrophages through TLR4-Induced ROS Signaling and A20-Mediated NLRP3 Deubiquitination.

The extracellular protozoan parasite Giardia duodenalis is a well-known and important causative agent of diarrhea on a global scale. Macrophage pyroptosis has been recognized as an important innate immune effector mechanism against intracellular pathogens. Yet, the effects of noninvasive Giardia infection on macrophage pyroptosis and the associated molecular triggers and regulators remain poorly defined. Here we initially observed that NLRP3 inflammasome-mediated pyroptosis was activated in Giardia-treated macrophages, and inhibition of ROS, NLRP3, or caspase-1 could block GSDMD cleavage, IL-1ß, IL-18 and LDH release, and the cell viability reduction. We also confirmed that Giardia-induced NLRP3 inflammasome activation was involved in its K63 deubiquitination. Thus, six candidate deubiquitinases were screened, among which A20 was identified as an effective regulator. We then screened TLRs on macrophage membranes and found that upon stimulation TLR4 was tightly correlated to ROS enhancement, A20-mediated NLRP3 deubiquitination, and pyroptotic signaling. In addition, several Giardia-secreted proteins were predicted as trigger factors via secretome analysis, of which peptidyl-prolyl cis-trans isomerase B (PPIB) independently induced macrophage pyroptosis. This was similar to the findings from the trophozoite treatment, and also led to the TLR4-mediated activation of NLRP3 through K63 deubiquitination by A20. Collectively, the results of this study have significant implications for expanding our understanding of host defense mechanisms after infection with G. duodenalis.

Giardia duodenalis Induces Apoptosis in Intestinal Epithelial Cells via Reactive Oxygen Species-Mediated Mitochondrial Pathway In Vitro.

The intestinal protozoan parasite, Giardia duodenalis, infects a large number of people in the world annually. Giardia infection has been considered a negative effect on intestinal epithelial cell growth, while the underlying mechanisms remain to be explored. Here we evaluated reactive oxygen species (ROS) production and apoptotic events in Giardia trophozoites-stimulated Caco-2 cells via fluorescence microscopy, transmission electron microscopy, flow cytometry, western blot, and cell counting kit-8 analyses. The results showed that Giardia trophozoite treatment could induce lactate dehydrogenase release and Caco-2 cell apoptosis. The ROS levels were increased post treatment. The observed typical characteristics of mitochondria damage include significant swelling and degeneration of matrix and cristae. After trophozoite treatment, the level of Bax protein expression was increased, while Bcl-2 protein decreased. Trophozoite stimulation also led to reduction of mitochondrial membrane potential and release of cytochrome c from the mitochondria to the cytoplasm, and this process was accompanied by activation of caspase-9 and caspase-3 and poly (ADP-ribose) polymerase 1 cleavage. Pretreatment with N-acetyl-L-cysteine, a ROS inhibitor, reversed G. duodenalis-induced Caco-2 cell apoptosis. Taken together, we indicated that G. duodenalis could induce Caco-2 cell apoptosis through a ROS- and mitochondria-mediated caspase-dependent pathway. This study furthers our understanding of the cellular mechanism of the interaction between Giardia trophozoites and host cells.

Octreotide remits endoplasmic reticulum stress to reduce autophagy of intestinal epithelial cell line Caco-2 via upregulation of miR-101.

Octreotide (OCT) shows clinical efficacies in the treatment of liver cirrhosis complicated with gastrointestinal hemorrhage. Experiments were designed to investigate its function mechanism associated with endoplasmic reticulum stress (ERS)-induced autophagy and microRNA (miR). Protein associated with ERS and autophagy was detected by western blot. miR-101 was examined by qRT-PCR. Besides, miR-101 or G protein-coupled receptor 78 (GPR78)-silenced Caco-2 cells were established by transfection. Furthermore, western blot was used to determine TGF-beta activated kinase 1 (TAK1), AMPK, mTOR, p70S6K as well as their phosphorylated forms. Lipopolysaccharide (LPS) enforced the expression of GPR78. Besides, LPS triggered the production of Beclin-1 and LC3-II while mitigated the accumulation of p62. Then all these above results were reversed by OCT pretreatment. Moreover, miR-101 expression was downregulated by LPS while upregulated by OCT. Further, miR-101 knockdown strengthened ERS and promoted autophagy. GPR78 silence retarded autophagy process. In the end, OCT mitigated phosphorylation of TAK1, AMPK while enhanced the phosphorylated expression of mTOR and p70S6K in LPS-treated Caco-2 cells. The anti-autophagy property of OCT was mediated by miR-101-induced suppression of GPR78 in LPS-treated Caco-2 cells.

Brassinosteroids play a critical role in the regulation of pesticide metabolism in crop plants.

Pesticide residues in agricultural produce pose a threat to human health worldwide. Although the detoxification mechanisms for xenobiotics have been extensively studied in mammalian cells, information about the regulation network in plants remains elusive. Here we show that brassinosteroids (BRs), a class of natural plant hormones, decreased residues of common organophosphorus, organochlorine and carbamate pesticides by 30-70% on tomato, rice, tea, broccoli, cucumber, strawberry, and other plants when treated externally. Genome-wide microarray analysis showed that fungicide chlorothalonil (CHT) and BR co-upregulated 301 genes, including a set of detoxifying genes encoding cytochrome P450, oxidoreductase, hydrolase and transferase in tomato plants. The level of BRs was closely related to the respiratory burst oxidase 1 (RBOH1)-encoded NADPH oxides-dependent H2O2 production, glutathione biosynthesis and the redox homeostasis, and the activity of glutathione S-transferase (GST). Gene silencing treatments showed that BRs decreased pesticide residues in plants likely by promoting their metabolism through a signaling pathway involving BRs-induced H2O2 production and cellular redox change. Our study provided a novel approach for minimizing pesticide residues in crops by exploiting plants' own detoxification mechanisms.