Targeting Dectin-1 and or VISTA enhances anti-tumor immunity in melanoma but not colorectal cancer model.

PURPOSE: Acquired resistance to immune checkpoint blockers (ICBs) is a major barrier in cancer treatment, emphasizing the need for innovative strategies. Dectin-1 (gene Clec7a) is a C-type lectin receptor best known for its ability to recognize ß-glucan-rich structures in fungal cell walls. While Dectin-1 is expressed in myeloid cells and tumor cells, its significance in cancer remains the subject of controversy. METHODS: Using Celc7a-/- mice and curdlan administration to stimulate Dectin-1 signaling, we explored its impact. VISTA KO mice were employed to assess VISTA's role, and bulk RNAseq analyzed curdlan effects on neutrophils. RESULTS: Our findings reveal myeloid cells as primary Dectin-1 expressing cells in the tumor microenvironment (TME), displaying an activated phenotype. Strong Dectin-1 co-expression/co-localization with VISTA and PD-L1 in TME myeloid cells was observed. While Dectin-1 deletion lacked protective effects, curdlan stimulation significantly curtailed B16-F10 tumor progression. RNAseq and pathway analyses supported curdlan's role in triggering a cascade of events leading to increased production of pro-inflammatory mediators, potentially resulting in the recruitment and activation of immune cells. Moreover, we identified a heterogeneous subset of Dectin-1+ effector T cells in the TME. Similar to mice, human myeloid cells are the prominent cells expressing Dectin-1 in cancer patients. CONCLUSION: Our study proposes Dectin-1 as a potential adjunctive target with ICBs, orchestrating a comprehensive engagement of innate and adaptive immune responses in melanoma. This innovative approach holds promise for overcoming acquired resistance to ICBs in cancer treatment, offering avenues for further exploration and development.

The Role of Coinhibitory Receptors in B Cell Dysregulation in SARS-CoV-2-Infected Individuals with Severe Disease.

Severe SARS-CoV-2 infection is associated with significant immune dysregulation involving different immune cell subsets. In this study, when analyzing critically ill COVID-19 patients versus those with mild disease, we observed a significant reduction in total and memory B cell subsets but an increase in naive B cells. Moreover, B cells from COVID-19 patients displayed impaired effector functions, evidenced by diminished proliferative capacity, reduced cytokine, and Ab production. This functional impairment was accompanied by an increased apoptotic potential upon stimulation in B cells from severely ill COVID-19 patients. Our further studies revealed the expansion of B cells expressing coinhibitory molecules (PD-1, PD-L1, TIM-1, VISTA, CTLA-4, and Gal-9) in intensive care unit (ICU)-admitted patients but not in those with mild disease. The coinhibitory receptor expression was linked to altered IgA and IgG expression and increased the apoptotic capacity of B cells. Also, we found a reduced frequency of CD24hiCD38hi regulatory B cells with impaired IL-10 production. Our mechanistic studies revealed that the upregulation of PD-L1 was linked to elevated plasma IL-6 levels in COVID-19 patients. This implies a connection between the cytokine storm and altered B cell phenotype and function. Finally, our metabolomic analysis showed a significant reduction in tryptophan but elevation of kynurenine in ICU-admitted COVID-19 patients. We found that kynurenine promotes PD-L1 expression in B cells, correlating with increased IL-6R expression and STAT1/STAT3 activation. Our observations provide novel insights into the complex interplay of B cell dysregulation, implicating coinhibitory receptors, IL-6, and kynurenine in impaired B cell effector functions, potentially contributing to the pathogenesis of COVID-19.

Metabolomic and immune alterations in long COVID patients with chronic fatigue syndrome.

Introduction: A group of SARS-CoV-2 infected individuals present lingering symptoms, defined as long COVID (LC), that may last months or years post the onset of acute disease. A portion of LC patients have symptoms similar to myalgic encephalomyelitis or chronic fatigue syndrome (ME/CFS), which results in a substantial reduction in their quality of life. A better understanding of the pathophysiology of LC, in particular, ME/CFS is urgently needed. Methods: We identified and studied metabolites and soluble biomarkers in plasma from LC individuals mainly exhibiting ME/CFS compared to age-sex-matched recovered individuals (R) without LC, acute COVID-19 patients (A), and to SARS-CoV-2 unexposed healthy individuals (HC). Results: Through these analyses, we identified alterations in several metabolomic pathways in LC vs other groups. Plasma metabolomics analysis showed that LC differed from the R and HC groups. Of note, the R group also exhibited a different metabolomic profile than HC. Moreover, we observed a significant elevation in the plasma pro-inflammatory biomarkers (e.g. IL-1α, IL-6, TNF-α, Flt-1, and sCD14) but the reduction in ATP in LC patients. Our results demonstrate that LC patients exhibit persistent metabolomic abnormalities 12 months after the acute COVID-19 disease. Of note, such metabolomic alterations can be observed in the R group 12 months after the acute disease. Hence, the metabolomic recovery period for infected individuals with SARS-CoV-2 might be long-lasting. In particular, we found a significant reduction in sarcosine and serine concentrations in LC patients, which was inversely correlated with depression, anxiety, and cognitive dysfunction scores. Conclusion: Our study findings provide a comprehensive metabolomic knowledge base and other soluble biomarkers for a better understanding of the pathophysiology of LC and suggests sarcosine and serine supplementations might have potential therapeutic implications in LC patients. Finally, our study reveals that LC disproportionally affects females more than males, as evidenced by nearly 70% of our LC patients being female.

Proof of concept: Pull down assay using bovine serum albumin and an immunomodulator small molecule.

In the past decade, there has been increasing interest in use of small molecules for immunomodulation. The affinity-based pull-down purification is an essential tool for target identification of small molecules and drug discovery. This study presents our recent efforts to investigate the cellular target(s) of Compound A, a small molecule with demonstrated immunomodulatory properties in human peripheral blood mononuclear cells (PBMCs). While we have previously observed the immunomodulatory activity of Compound A in PBMCs, the specific molecular targets underlying its effects remains elusive. To address this challenge, we synthesized a trifluoromethyl phenyl diazirine (TPD)-bearing trifunctional Probe 1 based on the chemical structure of Compound A, which could be used in a pull-down assay to efficiently bind to putative cellular targets via photoaffinity labelling. In this report, we utilized bovine serum albumin (BSA) as a model protein to establish a proof-of-concept in order to assess the suitability of Probe 1 for binding to an endogenous target. By the successful synthesis of Probe 1 and demonstrating the efficient binding of Probe 1 to BSA, we propose that this method can be used as a tool for further identification of potential protein targets of small molecules in living cells. Our findings provide a valuable starting point for further investigations into the molecular mechanisms underlying the immunomodulatory effects of Compound A.

Immunological responses in SARS-CoV-2 and HIV co-infection versus SARS-CoV-2 mono-infection: case report of the interplay between SARS-CoV-2 and HIV.

BACKGROUND: There is an urgent need to understand the interplay between SARS-CoV-2 and HIV to inform risk-mitigation approaches for HIV-infected individuals. OBJECTIVES: We conclude that people living with HIV (PLWH) who are antiretroviral therapy (ART) naïve could be at a greater risk of morbidity or mortality once co-infected with SARS-CoV-2. METHODS: Here, we performed extensive immune phenotyping using flow cytometry. Moreover, to compare the range of values observed in the co-infected case, we have included a larger number of mono-infected cases with SARS-CoV-2. We also quantified soluble co-inhibitory/co-stimulatory molecules in the plasma of our patients. RESULTS: We noted a robust immune activation characterized by the expansion of CD8+ T cells expressing co-inhibitory/stimulatory molecules (e.g. PD-1, TIM-3, 2B4, TIGIT, CD39, and ICOS) and activation markers (CD38, CD71, and HLA-DR) in the co-infected case. We further found that neutrophilia was more pronounced at the expense of lymphopenia in the co-infected case. In particular, naïve and central memory CD8+ T cells were scarce as a result of switching to effector and effector memory in the co-infected case. CD8+ T cell effector functions such as cytokine production (e.g. TNF-α and IFN-γ) and cytolytic molecules expression (granzyme B and perforin) following anti-CD3/CD28 or the Spike peptide pool stimulation were more prominent in the co-infected case versus the mono-infected case. We also observed that SARS-CoV-2 alters T cell exhaustion commonly observed in PLWH. CONCLUSION: These findings imply that inadequate immune reconstitution and/or lack of access to ART could dysregulate immune response against SARS-CoV-2 infection, which can result in poor clinical outcomes in PLWH. Our study has implications for prioritizing PLWH in the vaccination program/access to ART in resource-constrained settings.

Analysis of SARS-CoV-2 isolates, namely the Wuhan strain, Delta variant, and Omicron variant, identifies differential immune profiles.

There is an urgent need to better understand the impact of different severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants on immune response and disease dynamics to facilitate better intervention strategies. Here, we show that SARS-CoV-2 variants differentially affect host immune responses. The magnitude and quantity of cytokines and chemokines were comparable in those infected with the Wuhan strain and the Delta variant. However, individuals infected with the Omicron variant had significantly lower levels of these mediators. We also found an elevation of plasma galectins (Gal-3, Gal-8, and Gal-9) in infected individuals, in particular, in those with the original strain. Soluble galectins exert a proinflammatory role in COVID-19 pathogenesis. This was illustrated by their correlation with the plasma levels of sCD14, sCD163, enhanced TNF-α/IL-6 secretion, and increased SARS-CoV-2 infectivity in vitro. Moreover, we observed enhanced CD4+ and CD8+ T cell activation in Wuhan strain-infected individuals. Surprisingly, there was a more pronounced T cell activation in those infected with the Omicron in comparison to the Delta variant. In line with T cell activation status, we observed a more pronounced expansion of T cells expressing different co-inhibitory receptors in patients infected with the Wuhan strain, followed by the Omicron and Delta variants. Individuals infected with the Wuhan strain or the Omicron variant had a similar pattern of plasma soluble immune checkpoints. Our results imply that a milder innate immune response might be beneficial and protective in those infected with the Omicron variant. Our results provide a novel insight into the differential impact of SARS-CoV-2 variants on host immunity. IMPORTANCE There is a need to better understand how different SARS-CoV-2 variants influence the immune system and disease dynamics to facilitate the development of better vaccines and therapies. We compared immune responses in 140 SARS-CoV-2-infected individuals with the Wuhan strain, the Delta variant, or the Omicron variant. All these patients were admitted to the intensive care unit and were SARS-CoV-2 vaccination naïve. We found that SARS-CoV-2 variants differentially affect the host immune response. This was done by measuring soluble biomarkers in their plasma and examining different immune cells. Overall, we found that the magnitude of cytokine storm in individuals infected with the Wuhan strain or the Delta variant was greater than in those infected with the Omicron variant. In light of enhanced cytokine release syndrome in individuals infected with the Wuhan strain or the Delta variant, we believe that a milder innate immune response might be beneficial and protective in those infected with the Omicron variant.

Transcriptomic profiling of peripheral blood cells in HPV-associated carcinoma patients receiving combined valproic acid and avelumab.

Human papillomavirus (HPV)-associated cancer continues to evade the immune system by promoting a suppressive tumor microenvironment. Therefore, immunotherapy appears to be a promising approach for targeting HPV-associated tumors. We hypothesized that valproic acid (VA) as an epigenetic agent combined with avelumab may enhance the antitumor immunity in HPV-associated solid tumors. We performed bulk RNA-sequencing (RNA-Seq) on total peripheral blood mononuclear cells (PBMCs) of seven nonresponders (NRs) and four responders (Rs). A total of 39 samples (e.g., pretreatment, post-VA, postavelumab, and endpoint) were analyzed. Also, we quantified plasma analytes and performed flow cytometry. We observed a differential pattern in immune response following treatment with VA and/or avelumab in NRs vs. Rs. A significant upregulation of transcripts associated with NETosis [the formation of neutrophil extracellular traps (NETs)] and neutrophil degranulation pathways was linked to the presence of a myeloid-derived suppressor cell signature in NRs. We noted the elevation of IL-8/IL-18 cytokines and a distinct transcriptome signature at the baseline and endpoint in NRs. By using the receiver operator characteristics, we identified a cutoff value for the plasma IL-8/IL-18 to discriminate NRs from Rs. We found differential therapeutic effects for VA and avelumab in NRs vs. Rs. Thus, our results imply that measuring the plasma IL-8/IL-18 and bulk RNA-Seq of PBMCs may serve as valuable biomarkers to predict immunotherapy outcomes.

SARS-CoV-2 Mpro Protease Variants of Concern Display Altered Viral Substrate and Cell Host Target Galectin-8 Processing but Retain Sensitivity toward Antivirals.

The main protease of SARS-CoV-2 (Mpro) is the most promising drug target against coronaviruses due to its essential role in virus replication. With newly emerging variants there is a concern that mutations in Mpro may alter the structural and functional properties of protease and subsequently the potency of existing and potential antivirals. We explored the effect of 31 mutations belonging to 5 variants of concern (VOCs) on catalytic parameters and substrate specificity, which revealed changes in substrate binding and the rate of cleavage of a viral peptide. Crystal structures of 11 Mpro mutants provided structural insight into their altered functionality. Additionally, we show Mpro mutations influence proteolysis of an immunomodulatory host protein Galectin-8 (Gal-8) and a subsequent significant decrease in cytokine secretion, providing evidence for alterations in the escape of host-antiviral mechanisms. Accordingly, mutations associated with the Gamma VOC and highly virulent Delta VOC resulted in a significant increase in Gal-8 cleavage. Importantly, IC50s of nirmatrelvir (Pfizer) and our irreversible inhibitor AVI-8053 demonstrated no changes in potency for both drugs for all mutants, suggesting Mpro will remain a high-priority antiviral drug candidate as SARS-CoV-2 evolves.

DNA methylation changes underlie the long-term association between periodontitis and atherosclerotic cardiovascular disease.

Periodontitis, the leading cause of adult tooth loss, has been identified as an independent risk factor for cardiovascular disease (CVD). Studies suggest that periodontitis, like other CVD risk factors, shows the persistence of increased CVD risk even after mitigation. We hypothesized that periodontitis induces epigenetic changes in hematopoietic stem cells in the bone marrow (BM), and such changes persist after the clinical elimination of the disease and underlie the increased CVD risk. We used a BM transplant approach to simulate the clinical elimination of periodontitis and the persistence of the hypothesized epigenetic reprogramming. Using the low-density lipoprotein receptor knockout (LDLRo ) atherosclerosis mouse model, BM donor mice were fed a high-fat diet to induce atherosclerosis and orally inoculated with Porphyromonas gingivalis (Pg), a keystone periodontal pathogen; the second group was sham-inoculated. Naïve LDLR o mice were irradiated and transplanted with BM from one of the two donor groups. Recipients of BM from Pg-inoculated donors developed significantly more atherosclerosis, accompanied by cytokine/chemokines that suggested BM progenitor cell mobilization and were associated with atherosclerosis and/or PD. Using whole-genome bisulfite sequencing, 375 differentially methylated regions (DMRs) and global hypomethylation in recipients of BM from Pg-inoculated donors were observed. Some DMRs pointed to the involvement of enzymes with major roles in DNA methylation and demethylation. In validation assays, we found a significant increase in the activity of ten-eleven translocase-2 and a decrease in the activity of DNA methyltransferases. Plasma S-adenosylhomocysteine levels were significantly higher, and the S-adenosylmethionine to S-adenosylhomocysteine ratio was decreased, both of which have been associated with CVD. These changes may be related to increased oxidative stress as a result of Pg infection. These data suggest a novel and paradigm-shifting mechanism in the long-term association between periodontitis and atherosclerotic CVD.

CD71+ erythroid cells suppress T-cell effector functions and predict immunotherapy outcomes in patients with virus-associated solid tumors.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of cancer. However, only a portion of patients respond to such treatments. Therefore, it remains a prevailing clinical need to identify factors associated with acquired resistance or lack of response to ICIs. We hypothesized that the immunosuppressive CD71+ erythroid cells (CECs) within the tumor and/or distant 'out-of-field' may impair antitumor response. METHODS: We studied 38 patients with cancer through a phase II clinical trial investigating the effects of oral valproate combined with avelumab (anti-programmed death-ligand 1 (PD-L1)) in virus-associated solid tumors (VASTs). We quantified the frequency/functionality of CECs in blood and biopsies of patients. Also, we established an animal model of melanoma (B16-F10) to investigate the possible effects of erythropoietin (EPO) treatment on anti-PD-L1 therapy. RESULTS: We found a substantial expansion of CECs in the blood of patients with VAST compared with healthy controls. We noted that the frequency of CECs in circulation was significantly higher at the baseline and throughout the study in non-responders versus responders to PD-L1 therapy. Moreover, we observed that CECs in a dose-dependent manner suppress effector functions of autologous T cells in vitro. The subpopulation of CD45+CECs appears to have a more robust immunosuppressive property compared with their CD45- counterparts. This was illustrated by a stronger expression of reactive oxygen species, PD-L1/PD-L2, and V-domain Ig suppressor of T-cell activation in this subpopulation. Lastly, we found a higher frequency of CECs in the blood circulation at the later cancer stage and their abundance was associated with anemia, and a poor response to immunotherapy. Finally, we report the expansion of CECs in the spleen and tumor microenvironment of mice with melanoma. We found that although CECs in tumor-bearing mice secret artemin, this was not the case for VAST-derived CECs in humans. Notably, our results imply that EPO, a frequently used drug for anemia treatment in patients with cancer, may promote the generation of CECs and subsequently abrogates the therapeutic effects of ICIs (eg, anti-PD-L1). CONCLUSIONS: Our results demonstrate that anemia by the expansion of CECs may enhance cancer progression. Notably, measuring the frequency of CECs may serve as a valuable biomarker to predict immunotherapy outcomes.

Galectin-9 promotes natural killer cells activity via interaction with CD44.

Natural killer (NK) cells are a potent innate source of cytokines and cytoplasmic granules. Their effector functions are tightly synchronized by the balance between the stimulatory and inhibitory receptors. Here, we quantified the proportion of NK cells and the surface presence of Galectin-9 (Gal-9) from the bone marrow, blood, liver, spleen, and lungs of adult and neonatal mice. We also examined the effector functions of Gal-9+NK cells compared with their Gal-9- counterparts. Our results revealed that Gal-9+NK cells are more abundant in tissues, in particular, in the liver than in the blood and bone marrow. We found Gal-9 presence was associated with enhanced cytotoxic effector molecules granzyme B (GzmB) and perforin expression. Likewise, Gal-9 expressing NK cells displayed greater IFN-γ and TNF-α expression than their negative counterparts under hemostatic circumstances. Notably, the expansion of Gal-9+NK cells in the spleen of mice infected with E. coli implies that Gal-9+NK cells may provide a protective role against infection. Similarly, we found the expansion of Gal-9+NK cells in the spleen and tumor tissues of melanoma B16-F10 mice. Mechanistically, our results revealed the interaction of Gal-9 with CD44 as noted by their co-expression/co-localization. Subsequently, this interaction resulted in enhanced expression of Phospho-LCK, ERK, Akt, MAPK, and mTOR in NK cells. Moreover, we found Gal-9+NK cells exhibited an activated phenotype as evidenced by increased CD69, CD25, and Sca-1 but reduced KLRG1 expression. Likewise, we found Gal-9 preferentially interacts with CD44high in human NK cells. Despite this interaction, we noted a dichotomy in terms of effector functions in NK cells from COVID-19 patients. We observed that the presence of Gal-9 on NK cells resulted in a greater IFN-γ expression without any changes in cytolytic molecule expression in these patients. These observations suggest differences in Gal-9+NK cell effector functions between mice and humans that should be considered in different physiological and pathological conditions. Therefore, our results highlight the important role of Gal-9 via CD44 in NK cell activation, which suggests Gal-9 is a potential new avenue for the development of therapeutic approaches to modulate NK cell effector functions.

Depletion of polyfunctional CD26highCD8+ T cells repertoire in chronic lymphocytic leukemia.

BACKGROUND: CD8+ T cells play an essential role against tumors but the role of human CD8+CD26+ T cell subset against tumors, in particular, haematological cancers such as chronic lymphocytic leukemia (CLL) remains unknown. Although CD4+CD26high T cells are considered for adoptive cancer immunotherapy, the role of CD8+CD26+ T cells is ill-defined. Therefore, further studies are required to better determine the role of CD8+CD26+ T cells in solid tumors and haematological cancers. METHODS: We studied 55 CLL and 44 age-sex-matched healthy controls (HCs). The expression of CD26 on different T cell subsets (e.g. naïve, memory, effector, and etc.) was analyzed. Also, functional properties of CD8+CD26+ and CD8+CD26- T cells were evaluated. Finally, the plasma cytokine/chemokine and Galectin-9 (Gal-9) levels were examined. RESULTS: CD26 expression identifies three CD8+ T cell subsets with distinct immunological properties. While CD26negCD8+ T cells are mainly transitional, effector memory and effectors, CD26lowCD8+ T cells are mainly naïve, stem cell, and central memory but CD26high T cells are differentiated to transitional and effector memory. CD26+CD8+ T cells are significantly reduced in CLL patients versus HCs. CD26high cells are enriched with Mucosal Associated Invariant T (MAIT) cells co-expressing CD161TVα7.2 and IL-18Rα. Also, CD26high cells have a rich chemokine receptor profile (e.g. CCR5 and CCR6), profound cytokine (TNF-α, IFN-γ, and IL-2), and cytolytic molecules (Granzyme B, K, and perforin) expression upon stimulation. CD26high and CD26low T cells exhibit significantly lower frequencies of CD160, 2B4, TIGIT, ICOS, CD39, and PD-1 but higher levels of CD27, CD28, and CD73 versus CD26neg cells. To understand the mechanism linked to CD26high depletion, we found that malignant B cells by shedding Galectin-9 (Gal-9) contribute to the elevation of plasma Gal-9 in CLL patients. In turn, Gal-9 and the inflammatory milieu (IL-18, IL-12, and IL-15) in CLL patients contribute to increased apoptosis of CD26high T cells. CONCLUSIONS: Our results demonstrate that CD26+ T cells possess a natural polyfunctionality to traffic and exhibit effector functions and resist exhaustion. Therefore, they can be proposed for adoptive cancer immunotherapy. Finally, neutralizing and/or inhibiting Gal-9 may preserve CD26highCD8+ T cells in CLL.

Differential effects of age, sex and dexamethasone therapy on ACE2/TMPRSS2 expression and susceptibility to SARS-CoV-2 infection.

ACE2 and TMPRSS2 are crucial for SARS-CoV-2 entry into the cell. Although ACE2 facilitates viral entry, its loss leads to promoting the devastating clinical symptoms of COVID-19 disease. Thus, enhanced ACE2/TMPRSS2 expression is likely to increase predisposition of target cells to SARS-CoV-2 infection. However, little evidence existed about the biological kinetics of these two enzymes and whether dexamethasone treatment modulates their expression. Here, we show that the expression of ACE2 at the protein and mRNA levels was significantly higher in the lung and heart tissues of neonatal compared to adult mice. However, the expression of TMPRSS2 was developmentally regulated. Our results may introduce a novel concept for the reduced susceptibility of the young to SARS-CoV-2 infection. Moreover, ACE2 expression but not TMPRSS2 was upregulated in adult female lungs compared to their male counterparts. Interestingly, the ACE2 and TMPRSS2 expressions were upregulated by dexamethasone treatment in the lung and heart tissues in both neonatal and adult mice. Furthermore, our findings provide a novel mechanism for the observed differential therapeutic effects of dexamethasone in COVID-19 patients. As such, dexamethasone exhibits different therapeutic effects depending on the disease stage. This was supported by increased ACE2/TMPRSS2 expression and subsequently enhanced infection of normal human bronchial epithelial cells (NHBE) and Vero E6 cells with SARS-CoV-2 once pre-treated with dexamethasone. Therefore, our results suggest that individuals who take dexamethasone for other clinical conditions may become more prone to SARS-CoV-2 infection.

Elevated IL-6 and IL-22 in Early Pregnancy Are Associated with Worse Disease Course in Women with Inflammatory Bowel Disease.

Inflammatory bowel diseases (IBD), including Ulcerative Colitis (UC) and Crohn's disease (CD), are inflammatory conditions of the intestinal tract that affect women in their reproductive years. Pregnancy affects Th1- and Th2-cytokines, but how these changes occur during pregnancy in IBD is unclear. We performed a longitudinal profiling of serum cytokines in a cohort of 11 healthy pregnant women and 76 pregnant women with IBD from the first trimester of pregnancy to the first 12 months post-partum. Participants were monitored for biochemical disease activity (C-reactive protein [CRP] and fecal calprotectin [FCP]) and clinical activities. Maternal cytokines were measured using ELISA. We identified changes in Th1 and Th17 cytokines throughout pregnancy in healthy pregnant women. During pregnancy, maternal serum cytokine expressions were influenced by IBD, disease activity, and medications. Active UC was associated with an elevation in IL-21, whereas active CD was associated with elevated IFN-γ, IL-6, and IL-21. Interestingly, T1 serum cytokine levels of IL-22 (>0.624 pg/mL) and IL-6 (>0.648 pg/mL) were associated with worse IBD disease activity throughout pregnancy in women with UC and CD, respectively. This shows serum cytokines in pregnancy differ by IBD, disease activity, and medications. We show for the first time that T1 IL-22 and IL-6 correlate with IBD disease course throughout pregnancy.

Differential Impact of SARS-CoV-2 Isolates, Namely, the Wuhan Strain, Delta, and Omicron Variants on Erythropoiesis.

SARS-CoV-2 variants exhibit different viral transmissibility and disease severity. However, their impact on erythropoiesis has not been investigated. Here, we show SARS-CoV-2 variants differentially affect erythropoiesis. This is illustrated by the abundance of CD71+ erythroid cells (CECs) in the blood circulation of COVID-19 patients infected with the original Wuhan strain followed by the Delta and Omicron variants. We observed the CD45+CECs are the dominant subpopulation of CECs expressing the receptor, ACE2, and coreceptor, TMPRSS2, and thus, can be targeted by SARS-CoV-2. Also, we found CECs exhibit immunosuppressive properties, specifically CD45+CECs are the dominant immunosuppressive cells and via reactive oxygen species (ROS) and arginase I expression can impair CD8+ T cell functions. In agreement, we observed CECs suppress CD8+ T cell effector (e.g., Granzyme B expression and degranulation capacity [CD107]), which was partially but significantly reversed with l-arginine supplementation. In light of the enriched frequency of CECs, in particular, CD45+CECs in patients infected with the original (Wuhan) strain, we believe this strain has a more prominent impact on hematopoiesis compared with the Delta and Omicron variants. Therefore, our study provides an important insight into the differential impact of SARS-CoV-2 variants on erythropoiesis in COVID-19 patients. IMPORTANCE Silent hypoxia has been the hallmark of SARS-CoV-2 infection. Red blood cells (RBCs) work as gas cargo delivering oxygen to different tissues. However, their immature counterparts reside in the bone marrow and normally absent in the blood circulation. We show SARS-CoV-2 infection is associated with the emergence of immature RBCs so called CD71+ erythroid cells (CECs) in the blood. In particular, we found these cells were more prevalent in the blood of those infected with the SARS-CoV-2 original strain (Wuhan) followed by the Delta and Omicron variants. This suggests SARS-CoV-2 directly or indirectly impacts RBC production. In agreement, we observed immature RBCs express the receptor (ACE2) and coreceptor (TMPRSS2) for SARS-CoV-2. CECs suppress T cells functions (e.g., Granzyme B and degranulation capacity) in vitro. Therefore, our study provides a novel insight into the differential impact of SARS-CoV-2 variants on erythropoiesis and subsequently the hypoxia commonly observed in COVID-19 patients.

Glucocorticoids Bind to SARS-CoV-2 S1 at Multiple Sites Causing Cooperative Inhibition of SARS-CoV-2 S1 Interaction With ACE2.

Dexamethasone may reduce mortality in COVID-19 patients. Whether dexamethasone or endogenous glucocorticoids, such as cortisol, biochemically interact with SARS-CoV-2 spike 1 protein (S1), or its cellular receptor ACE2, is unknown. Using molecular dynamics (MD) simulations and binding energy calculations, we identified 162 druggable pockets in various conformational states of S1 and all possible binding pockets for cortisol and dexamethasone. Through biochemical binding studies, we confirmed that cortisol and dexamethasone bind to S1. Limited proteolysis and mass spectrometry analyses validated several MD identified binding pockets for cortisol and dexamethasone on S1. Interaction assays indicated that cortisol and dexamethasone separately and cooperatively disrupt S1 interaction with ACE2, through direct binding to S1, without affecting ACE2 catalytic activity. Cortisol disrupted the binding of the mutant S1 Beta variant (E484K, K417N, N501Y) to ACE2. Delta and Omicron variants are mutated in or near identified cortisol-binding pockets in S1, which may affect cortisol binding to them. In the presence of cortisol, we find increased inhibition of S1 binding to ACE2 by an anti-SARS-CoV-2 S1 human chimeric monoclonal antibody against the receptor binding domain. Whether glucocorticoid/S1 direct interaction is an innate defence mechanism that may have contributed to mild or asymptomatic SARS-CoV-2 infection deserves further investigation.

Hematopoietic responses to SARS-CoV-2 infection.

Under physiological conditions, hematopoietic stem and progenitor cells (HSPCs) in the bone marrow niches are responsible for the highly regulated and interconnected hematopoiesis process. At the same time, they must recognize potential threats and respond promptly to protect the host. A wide spectrum of microbial agents/products and the consequences of infection-induced mediators (e.g. cytokines, chemokines, and growth factors) can have prominent impact on HSPCs. While COVID-19 starts as a respiratory tract infection, it is considered a systemic disease which profoundly alters the hematopoietic system. Lymphopenia, neutrophilia, thrombocytopenia, and stress erythropoiesis are the hallmark of SARS-CoV-2 infection. Moreover, thrombocytopenia and blood hypercoagulability are common among COVID-19 patients with severe disease. Notably, the invasion of erythroid precursors and progenitors by SARS-CoV-2 is a cardinal feature of COVID-19 disease which may in part explain the mechanism underlying hypoxia. These pieces of evidence support the notion of skewed steady-state hematopoiesis to stress hematopoiesis following SARS-CoV-2 infection. The functional consequences of these alterations depend on the magnitude of the effect, which launches a unique hematopoietic response that is associated with increased myeloid at the expense of decreased lymphoid cells. This article reviews some of the key pathways including the infectious and inflammatory processes that control hematopoiesis, followed by a comprehensive review that summarizes the latest evidence and discusses how SARS-CoV-2 infection impacts hematopoiesis.

Elevated ATP via enhanced miRNA-30b, 30c, and 30e downregulates the expression of CD73 in CD8+ T cells of HIV-infected individuals.

CD8+ T cells play a crucial role against chronic viral infections, however, their effector functions are influenced by the expression of co-stimulatory/inhibitory receptors. For example, CD73 works with CD39 to convert highly inflammatory ATP to adenosine. However, its expression on T cells in the context of viral infections has not been well defined. Here, we analyzed the expression of CD73 on human T cells in a cohort of 102 HIV-infected individuals including those on antiretroviral therapy (ART), ART-naïve, and long-term non-progressors who were not on ART. We found that the frequency of CD73+ T cells was markedly lower among T cell subsets (e.g. naïve, effector or memory) in the peripheral blood of all HIV-infected individuals. Notably, CD73 was decreased at the cell surface, intracellular and gene levels. Functionally, CD8+CD73+ T cells exhibited decreased cytokine expression (TNF-α, IFN-γ and IL-2) upon global or antigen-specific stimulation and impaired expression of cytolytic molecules at the gene and protein levels. In contrast, CD8+CD73+ T cells expressed elevated levels of homing receptors such as CCR7, α4ß7 integrin, which suggests a migratory advantage for these cells as observed in vitro. We also observed significant migration of CD73+CD8+ T cells into the cerebrospinal fluids of multiple sclerosis (MS) patients at the time of disease relapse. Moreover, we found that elevated levels of ATP in the plasma of HIV-infected individuals upregulates the expression of miRNA30b-e in T cells in vitro. In turn, inhibition of miRNAs (30b, 30c and 30e) resulted in significant upregulation of CD73 mRNA in CD8+ T cells. Therefore, we provide a novel mechanism for the downregulation of CD73 via ATP-induced upregulation of miRNA30b, 30c and 30e in HIV infection. Finally, these observations imply that ATP-mediated downregulation of CD73 mainly occurs via its receptor, P2X1/P2RX1. Our results may in part explain why HIV-infected individuals have reduced risk of developing MS considering the role of CD73 for efficient T cell entry into the central nervous system.

Metagenomics Versus Metatranscriptomics of the Murine Gut Microbiome for Assessing Microbial Metabolism During Inflammation.

Shotgun metagenomics studies have improved our understanding of microbial population dynamics and have revealed significant contributions of microbes to gut homeostasis. They also allow in silico inference of the metagenome. While they link the microbiome with metabolic abnormalities associated with disease phenotypes, they do not capture microbial gene expression patterns that occur in response to the multitude of stimuli that constantly ambush the gut environment. Metatranscriptomics closes that gap, but its implementation is more expensive and tedious. We assessed the metabolic perturbations associated with gut inflammation using shotgun metagenomics and metatranscriptomics. Shotgun metagenomics detected changes in abundance of bacterial taxa known to be SCFA producers, which favors gut homeostasis. Bacteria in the phylum Firmicutes were found at decreased abundance, while those in phyla Bacteroidetes and Proteobacteria were found at increased abundance. Surprisingly, inferring the coding capacity of the microbiome from shotgun metagenomics data did not result in any statistically significant difference, suggesting functional redundancy in the microbiome or poor resolution of shotgun metagenomics data to profile bacterial pathways, especially when sequencing is not very deep. Obviously, the ability of metatranscriptomics libraries to detect transcripts expressed at basal (or simply low) levels is also dependent on sequencing depth. Nevertheless, metatranscriptomics informed about contrasting roles of bacteria during inflammation. Functions involved in nutrient transport, immune suppression and regulation of tissue damage were dramatically upregulated, perhaps contributed by homeostasis-promoting bacteria. Functions ostensibly increasing bacteria pathogenesis were also found upregulated, perhaps as a consequence of increased abundance of Proteobacteria. Bacterial protein synthesis appeared downregulated. In summary, shotgun metagenomics was useful to profile bacterial population composition and taxa relative abundance, but did not inform about differential gene content associated with inflammation. Metatranscriptomics was more robust for capturing bacterial metabolism in real time. Although both approaches are complementary, it is often not possible to apply them in parallel. We hope our data will help researchers to decide which approach is more appropriate for the study of different aspects of the microbiome.

Post-neonatal Outcomes of Infants Born to Women with Active Trimester One Inflammatory Bowel Disease: A Pilot Study.

BACKGROUND: Ulcerative colitis (UC) and Crohn's disease (CD) are chronic inflammatory bowel diseases (IBD) that affect women in their childbearing years. Early pregnancy flare-up negatively impacts obstetrical and perinatal outcomes, but the impact on infants is unclear. AIM: To determine whether active IBD disease activity is associated with adverse post-neonatal outcomes post-partum. METHODS: This is a single-center cohort study of women with IBD who underwent serial monitoring of post-neonatal outcomes post-partum. Infant outcomes were collected via self-filled questionnaires, including perinatal outcomes, APGAR scores, infant weights, heights, feeding habits and comorbidities within the first year of life. RESULTS: There was a total of 98 women with IBD and 78 live births throughout the study: 50 women were enrolled during trimester one alone and 49 were included into the current study. Among the 49 analyzed, 32 were in remission and 17 were in relapse during trimester one. Trimester one disease activity was associated with more adverse obstetrical outcomes including emergency C-sections and reduced 1-min APGAR scores. At follow-up, infants born to women with T1-flare had reduced weight-for-age Z scores and length-for-age Z scores up to 6 months of age. CONCLUSIONS: Active IBD during trimester one is correlated with adverse post-neonatal outcomes, particularly decreased infant weight and height up to 6 months of age. This suggests disease control in first trimester is essential for optimizing infant growth and post-neonatal outcomes.