Alpha 1-acid glycoprotein is upregulated in severe COVID-19 patients and decreases neutrophil NETs in SARS-CoV-2 infection.

Orosomucoid, or alpha-1 acid glycoprotein (AGP), is a major acute-phase protein expressed in response to systemic injury and inflammation. AGP has been described as an inhibitor of neutrophil migration on sepsis, particularly its immunomodulation effects. AGP's biological functions in coronavirus disease 2019 (COVID-19) are not understood. We sought to investigate the role of AGP in severe COVID-19 infection patients and neutrophils infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Epidemiological data, AGP levels, and other laboratory parameters were measured in blood samples from 56 subjects hospitalized in the ICU with SARS-CoV-2 infection. To evaluate the role of AGP in NETosis in neutrophils, blood samples from health patients were collected, and neutrophils were separated and infected with SARS-CoV-2. Those neutrophils were treated with AGP or vehicle, and NETosis was analyzed by flow cytometry. AGP was upregulated in severe COVID-19 patients (p<0.05). AGP level was positively correlated with IL-6 and C-reactive protein (respectively, p=0.005, p=0.002) and negatively correlated with lactate (p=0.004). AGP treatment downregulated early and late NETosis (respectively, 35.7% and 43.5%) in neutrophils infected with SARS-CoV-2 and up-regulated IL-6 supernatant culture expression (p<0.0001). Our data showed increased AGP in COVID-19 infection and contributed to NETosis regulation and increased IL-6 production, possibly related to the Cytokine storm in COVID-19.

Good cops turn bad: The contribution of neutrophils to immune-checkpoint inhibitor treatment failures in cancer.

Immune checkpoint inhibitor therapy activates tumor-killing T-cells by releasing the brake of anti-tumor immunity. It has been approved as first- or second-line therapy in many cancer types. Unfortunately, a majority of immune checkpoint inhibitor recipients are refractory to the therapy. Recent investigations of the peripheral blood and tumor microenvironment of cancer patients indicate that high neutrophil content is associated with poor response rates, suggesting an opportunity for synergistic therapy. In the current review, we discuss the mechanisms of neutrophil-mediated immunosuppression in cancer and recent findings suggesting that neutrophil antagonism will improve the efficacy of immune checkpoint inhibitor therapy.

Induction and Regulation of the Immunoproteasome Subunit ß5i (PSMB8) in Laryngeal and Hypopharyngeal Carcinoma Cells.

BACKGROUND The ubiquitin-proteasome pathway (UPP) is closely associated with the occurrence and progression of cancer, and the 5i immunoproteasome subunit is an important antitumor target in UPP. This study aimed to characterize the regulation of the immunoproteasome subunit ß5i (PSMB8) in JHU-011 laryngeal carcinoma cells and FaDu hypopharyngeal carcinoma cells to explore a new target for the treatment of laryngeal and hypopharyngeal carcinomas. MATERIAL AND METHODS JHU-011 and FaDu cells were used as effector cells in this study. By means of 6°Co γ-irradiation, the construction of stable cell lines of the silenced proto-oncogene c-Abl, and the addition of exogenous tyrosine kinase inhibitor (TKI) and activator, the transcription and protein expression levels of PSMB8 and its alternatively spliced isoforms in both cell lines were detected by real-time fluorescence quantitative polymerase chain reaction (RT-PCR) and Western blot. RESULTS Ionizing radiation upregulated the transcription level of the alternatively spliced isoform of PSMB8, E2, in both cell lines, thereby upregulating the mRNA and protein levels of PSMB8. The silencing of the proto-oncogene c-Abl and the activation and inhibition of its kinetic kinase product can affect the transcription and protein levels of PSMB8. CONCLUSIONS Ionizing radiation can significantly upregulate the mRNA and protein levels of PSMB8, which happens through the upregulation of its splicing isoform E2. The proto-oncogene c-Abl and its kinetic kinase protein product can regulate the transcription and protein expression levels of PSMB8 and its alternatively spliced isoforms.

The Immunoproteasome: An Emerging Target in Cancer and Autoimmune and Neurological Disorders.

The immunoproteasome (iCP) is an isoform of the 20S proteasome that is expressed when cells are stressed or receive an inflammatory signal. The primary role of the iCP is to hydrolyze proteins into peptides that are compatible with being loaded into a MHC-I complex. When the activity of the iCP is dysregulated or highly expressed, it can lead to unwanted cell death. Some cancer types express the iCP rather than the standard proteasome, and selective inhibitors have been developed to exploit this difference. Here, we describe diseases known to be influenced by iCP activity and the current status for targeting the iCP to elicit a therapeutic response. We also describe a variety of chemical tools that have been developed to monitor the activity of the iCP in cells. Finally, we present the future outlook for targeting the iCP in a variety of disease types and with mechanisms besides inhibition.

Immunoproteasome Subunit ß5i Promotes Ang II (Angiotensin II)-Induced Atrial Fibrillation by Targeting ATRAP (Ang II Type I Receptor-Associated Protein) Degradation in Mice.

Atrial fibrillation (AF) is the most common type of cardiac arrhythmia and increases the risk of stroke, heart failure, and death. Ang II (angiotensin II) triggers AF, mainly through stimulation of the AT1R (Ang II type I receptor). The immunoproteasome is a highly efficient proteolytic machine derived from the constitutive proteasome, but the role it plays in regulating AT1R activation and triggering AF remains unknown. Here, we show that among the catalytic subunits, ß5i (PSMB8) expression, and chymotrypsin-like activity were the most significantly upregulated in atrial tissue of Ang II-infused mice or serum from patients with AF. ß5i KO (ß5i knockout) in mice markedly attenuated Ang II-induced AF incidence, atrial fibrosis, inflammatory response, and oxidative stress compared with WT (wild type) animals, but injection with recombinant adeno-associated virus serotype 9-ß5i increased these effects. Moreover, we found that ATRAP (AT1R-associated protein) was a target of ß5i. Overexpression of ATRAP significantly attenuated Ang II-induced atrial remodeling and AF in recombinant adeno-associated virus serotype 9-ß5i-injected mice. Mechanistically, Ang II upregulated ß5i expression to promote ATRAP degradation, which resulted in activation of AT1R-mediated NF-κB signaling, increased NADPH oxidase activity, increased TGF (transforming growth factor)-ß1/Smad signaling, and altered the expression of Kir2.1 and CX43 (connexin 43) in the atria, thereby affecting atrial remodeling and AF. In summary, this study identifies ß5i as a negative regulator of ATRAP stability that contributes to AT1R activation and to AF, highlighting that targeting ß5i activity may represent a potential therapeutic approach for the treatment of hypertensive AF.

Plasma Immunoproteasome Predicts Early Hemorrhagic Transformation in Acute Ischemic Stroke Patients.

BACKGROUND AND PURPOSE: Currently, blood biomarkers associated with an increased hemorrhagic transformation (HT) risk remain uncertain. We aimed to determine the significance of immunoproteasome as predictors of early HT in acute ischemic stroke patients. METHODS: This study enrolled 316 patients with ischemic stroke. HT was assessed by computed tomography examination performed on day 5 ± 2 after stroke onset or immediately in case of clinical deterioration (CD). Plasma immunoproteasome subunits low molecular mass peptide 2 (LMP2), multicatalytic endopeptidase complex-like 1 (MECL-1), LMP7, interleukin-1ß (IL1ß), and high-sensitivity C-reactive protein (Hs-CRP) were measured with quantitative sandwich enzyme-linked immunosorbent assay kits. Factors associated with HT were analyzed using a multivariate logistic regression analysis. RESULTS: There were 42 (13.3%, 42 of 316) patients who experienced HT. Compared with those patients without HT, plasma LMP2, MECL-1, LMP7, IL1ß, and Hs-CRP concentrations on admission were significantly increased in patients with subsequent HT (P < .001). These protein concentrations increased with hemorrhage severity. Patients with CD caused by HT had the highest levels of LMP2 (1679.5 [1394.6-136.6] pg/mL), MECL-1 (992.5 [849.7-1075.8] pg/mL), LMP7 (822.6 [748.6-1009.5] pg/mL), IL1ß (113.2 [90.6-194.5] pg/mL), and Hs-CRP (30.0 [12.8-75.6] mg/L) (P < .05). Logistic regression analysis showed cardioembolism, LMP2, MECL-1, and LMP7 as independent predictors of HT (P < .05). Receiver operating characteristic curve analysis demonstrated LMP2 ≥ 988.3 pg/mL, MECL-1 ≥ 584.7 pg/mL, and LMP7 ≥ 509.0 pg/mL as independent factors associated with HT (P < .001). CONCLUSION: Evaluation of plasma levels of immunoproteasome could be helpful in the early prediction of HT in acute ischemic stroke patients.

Identification of immunodominant proteins from Mannheimia haemolytica and Histophilus somni by an immunoproteomic approach.

Mannheimia haemolytica and Histophilus somni are frequently isolated from diseased cattle with bovine respiratory disease (BRD). They compromise animal lung function and the immune responses generated are not sufficient to limit infection. Identification of specific immunogenic antigens for vaccine development represents a great challenge. Immunogenic proteins were identified by immunoproteomic approach with sera from cattle immunized with a commercial cellular vaccine of M. haemolytica and H. somni. Proteins of M. haemolytica were identified as solute ABC transporter, iron-binding protein, and hypothetical protein of capsular biosynthesis. Histophilus somni proteins correspond to porin, amino acid ABC transporter, hypothetical outer membrane protein, cysteine synthase, and outer membrane protein P6. Although these antigens share strong similarities with other proteins from animal pathogens, the ABC system proteins have been associated with virulence and these proteins could be considered as potential vaccine candidates for BRD.

Mannheimia haemolytica et Histophilus somni sont fréquemment isolées de bovins atteints de maladies respiratoires bovines (MRB). Ces agents compromettent la fonction pulmonaire et les réponses immunitaires générées ne permettent pas de limiter l'infection. L'identification d'antigènes spécifiques et immunogènes qui permettraient le développement de vaccins, représente un grand défi actuellement. Les protéines immunogènes ont été identifiées par une approche immunoproteomique en utilisant des sérums provenant de bovins immunisés par des vaccins commerciaux de M. haemolytica et H. somni. Les protéines de M. haemolytica ont été identifiées comme étant un transporteur ABC, une protéine de liaison du fer et une hypothétique protéine impliquée dans la biosynthèse de la capsule. Celles de H. somni correspondent à une porine, à un transporteur ABC d'acides aminés, à une hypothétique protéine de membrane externe, à la cystéine synthase et à la protéine membranaire P6. Bien que ces antigènes présentent une forte homologie avec des protéines provenant d'autres pathogènes d'animaux, les protéines du système ABC sont associées à la virulence et pourraient être considérées comme des candidats potentiels pour l'élaboration de vaccins contre les MBR.(Traduit par Docteur Patricia Dupre).

Alteration of immunoproteome profile of Echinococcus granulosus hydatid fluid with progression of cystic echinococcosis.

BACKGROUND: Cystic echinococcosis (CE), caused by Echinococcus granulosus metacestode, invokes a serious public health concern. Early diagnosis has great impacts on reduction of disability-adjusted life years. Several antigen B-related molecules (EgAgB; EgAgB1-5) are known to be immunopotent, but detection of EgAgB is variable in many patients and may not allow reliable interpretation of its immunological relevance. More importantly, the immunoproteome profile of hydatid fluid (HF) has not been addressed. METHODS: We conducted a proteome analysis of the HF of a single fertile cyst of CE1 and CE2 stages through two-dimensional electrophoresis (2-DE). Each protein spot was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). We subsequently determined the immunoproteome profile employing patient sera of entire disease spectrum from CE1 to CE5 stages. RESULTS: We identified 40 parasite proteins, of which EgAgB (28 spots) and antigen 5 (EgAg5; 5 molecules) were abundant. EgAgB proteoforms constituted the majority, mostly EgAgB1 (24 spots), followed by EgAgB2 and EgAgB4 (2 spots each). EgAgB3 was detected only by liquid chromatography-MS/MS. EgAgB5 was not recognized. We also detected 38 host proteins, which were largely composed of serum components, antioxidant/xenobiotic enzymes, and enzymes involved in carbohydrate metabolism. CE1 and CE2 HF exhibited comparable spotting patterns, but CE2 HF harbored greater amounts of EgAgB and EgAg5 complexes. CE sera demonstrated complicated immune recognition patterns according to the disease progression; CE2 and CE3 stages exhibited strong antibody responses against diverse EgAgB and EgAg5 proteoforms, while CE1, CE4, and CE5 stages mainly reacted to EgAg5 and cathepsin B. Patient sera of alveolar echinococcosis (AE) cross-reacted with diverse EgAgB isoforms (36%). EgAg5 and cathepsin B also demonstrated cross-reactions with sera from neurocysticercosis and sparganosis. CONCLUSIONS: Our results demonstrated that detection of a single defined molecule may not properly diagnose CE, since specific immunodominant epitopes changed as the disease progresses. Immunoproteome analysis combined with imaging studies may be practical in the differential diagnosis of CE from AE and other cystic lesions, as well as for staging CE, which are pertinent to establish appropriate patient management.

Immuno-histochemical determination of humoral immune markers within bacterial induced granuloma formation in Atlantic cod (Gadus morhua L.).

In this study the involvement of several humoral immune parameters of Atlantic cod (Gadus morhua L.) were studied in granuloma formed as a result of infection by Aeromonas salmonicida ssp. achomogenes. The results showed a clear association of immune parameters within the granuloma, in particular the localization of complement component C3, and including evidence for the presence of IgM, APoLP-A1 (Apolipoprotein), CRP-PI and CRP-PII (pentraxin).

The immunoproteasome in steatohepatitis: its role in Mallory-Denk body formation.

Recently it has been shown that the expression of the immunoproteasome increased in proportion to the degree of chronic inflammation in both the liver cell cytoplasm and nuclei in liver biopsies from patients who had chronic active hepatitis or cirrhosis. In the present study, biopsies from patients with steatohepatitis, with or without Mallory-Denk body (MDB) formation, were studied by immunofluorescent staining. Normal liver showed colocalization of FAT10, LMP2, LMP7, and MECL-1 at the mitochondria. Only LMP2 and LMP7 were found in the cell nuclei. Liver biopsies from patients with steatohepatitis and MDB formation, and a case of hepatocellular carcinoma forming MDBs in the tumor cells, showed colocalization of FAT10 and ubiquitin with LMP2, LMP7 and MECL-1 within the MDB. This indicates involvement of the immunoproteasome in MDB formation in steatohepatitis cases and in a case of HCC forming MDBs. Prior studies have shown that the immunoproteasome was involved in drug-induced MDB formation using the same immunofluorescent colocalization approach as was used on these human liver biopsies. The increase in the immunoproteasome subunit proteins was made at the expense of the 26S proteasome. This indicates that the shift from the 26S to the immunoproteasome had occurred in the MDB positive hepatocytes.

Proteomic analysis of the luteal endometrial secretome.

Endometrium attains a secretory architecture in preparation for embryo implantation, but the identity of most endometrial secretory products remains unknown. Our objective was to characterize the endometrial secretome and compare protein expression between prereceptive (luteinizing hormone [LH]+4) and receptive (LH+9) phase endometrium. Endometrial lavage was performed in 11 participants and analyzed by difference gel electrophoresis (DIGE). LH+4 and LH+9 specimens were labeled with cyanine fluorescent dyes Cy3 and Cy5 tags, respectively, and combined. Proteins were separated using 2-dimensional gel electrophoresis, isolated, trypsin-digested, and subjected to mass spectrometry. In all, 152 proteins were identified; 82 were differentially expressed. Most proteins with increased expression on LH+9 functioned in host defense, while proteins with decreased expression had many functions. A total of 14 proteins had changes suggesting altered posttranslational modification. This article describes the first application of proteomic analysis to endometrial secretions, allowing identification of novel endometrial proteins as well as those differentially secreted in prereceptive and receptive phases.

Activation of the human contact system on neutrophil extracellular traps.

Pattern recognition is an integral part of the innate immune system. The human contact system has been shown to interact with the surface of many bacterial and fungal pathogens, and once activated leads to the generation of antimicrobial peptides and the proinflammatory mediator bradykinin. Here we show that apart from these surfaces also neutrophil extracellular traps (NETs) provide a surface that allows the binding and activation of the contact system. In addition, we present evidence that M1 protein, a streptococcal surface protein, in concert with human fibrinogen triggers polymorphonuclear neutrophils to form NETs.

Cord immunoproteins as predictors of respiratory outcome in preterm infants.

OBJECTIVE: The purpose of this study was to evaluate the role of cord blood proteins and antenatal factors in the prediction of respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD). STUDY DESIGN: The prospectively collected cohort included 163 infants. All infants were born between 1998-2002 in a single regional hospital before 32 weeks of gestation and survived the first hospitalization. Altogether, 107 cord blood proteins were analyzed. Twenty-two antenatal clinical factors were included in the data mining and logistic regression analyses. RESULTS: The incidence of RDS was 64% and of BPD was 25%. Histologic chorioamnionitis protected from RDS (odds ratio [OR], 0.24; 95% confidence interval [CI], 0.11-0.53; P < .001). Besides the length of gestation, other clinical factors poorly predicted the outcomes. Matrix metalloproteinase-9 independently predicted RDS (OR, 8.3; 95% CI, 3.0-23.1; P < .001). Soluble glycoprotein 130 independently predicted BPD (OR, 6.07; 95%CI, 2.20-16.7; P < .001). CONCLUSION: Specific antenatal immunologic activation predicts either acute or chronic respiratory disease in very preterm infants.

Immunoproteasome in Macaca fascicularis: no age-dependent modification of abundance and activity in the brain and insight into an in silico structural model.

In this study, we investigated proteasome composition and activity in the brain of Macaca fascicularis, in order to test whether this nonhuman primate species might be a suitable animal model for anti-aging therapies in the central nervous system, addressed to the ubiquitin-proteasome system. We detected the catalytic beta subunits of constitutive proteasome, as well as the PA28 regulator and a subunit of immunoproteasome (i.e., beta1i [LMP2]), in seven adult, six old, and one young nonhuman primate brains. Subunit expression and proteasome activity were not influenced by the age of the animal in any of the brain regions (temporal and frontal cortex and cerebellum) we studied. However, an area-specific susceptibility to aged-related oxidative stress emerged. On the whole, the results suggest that, compared to humans, Macaca fascicularis primates may have a different age-dependent regulation of the ubiquitin-proteasome system and, possibly, of neuroinflammation in the brain. An in silico model of the 20S immunoproteasome containing the Macaca fascicularis alpha and beta subunits, present in database or identified by our group (i.e., LMP2), has been developed. Additional information was obtained by de novo sequencing of the beta1 (delta) subunit of Macaca fascicularis. A comparison with humans suggests that in multiprotein complexes some functional subunits, such as alpha subunits, appear to be preferentially conserved during evolution.