Dysfunctional purinergic signaling correlates with disease severity in COVID-19 patients

Ectonucleotidases modulate inflammatory responses by balancing extracellular ATP and adenosine (ADO) and might be involved in COVID-19 immunopathogenesis. Here, we explored the contribution of extracellular nucleotide metabolism to COVID-19 severity in mild and severe cases of the disease. We verified that the gene expression of ectonucleotidases is reduced in the whole blood of patients with COVID-19 and is negatively correlated to levels of CRP, an inflammatory marker of disease severity. In line with these findings, COVID-19 patients present higher ATP levels in plasma and reduced levels of ADO when compared to healthy controls. Cell type-specific analysis revealed higher frequencies of CD39+ T cells in severely ill patients, while CD4+ and CD8+ expressing CD73 are reduced in this same group. The frequency of B cells CD39+CD73+ is also decreased during acute COVID-19. Interestingly, B cells from COVID-19 patients showed a reduced capacity to hydrolyze ATP into ADP and ADO. Furthermore, impaired expression of ADO receptors and a compromised activation of its signaling pathway is observed in COVID-19 patients. The presence of ADO in vitro, however, suppressed inflammatory responses triggered in patients’ cells. In summary, our findings support the idea that alterations in the metabolism of extracellular purines contribute to immune dysregulation during COVID-19, possibly favoring disease severity, and suggest that ADO may be a therapeutic approach for the disease

Adjuvant Effect of Killed Propionibacterium acnes on Mouse Peritoneal B-1 Lymphocytes and Their Early Phagocyte Differentiation

B-1 lymphocytes are the predominant cells in mouse peritoneal cavity. They express macrophage and lymphocyte markers and are divided into B-1a, B-1b and B-1c subtypes. The role of B-1 cells is not completely clear, but they are responsible for natural IgM production and seem to play a regulatory role. An enriched B-1b cell population can be obtained from non-adherent peritoneal cell cultures, and we have previously demonstrated that these cells undergo differentiation to acquire a mononuclear phagocyte phenotype upon attachment to the substrate in vitro. Nevertheless, the B-1 cell response to antigens or adjuvants has been poorly investigated. Because killed Propionibacterium acnes exhibits immunomodulatory effects on both macrophages and B-2 lymphocytes, we analyzed whether a killed bacterial suspension or its soluble polysaccharide (PS) could modulate the absolute number of peritoneal B-1 cells in BALB/c mice, the activation status of these cells and their ability to differentiate into phagocytes in vitro. In vivo, P. acnes treatment elevated the absolute number of all B-1 subsets, whereas PS only increased B-1c. Moreover, the bacterium increased the number of B-1b cells that were positive for MHC II, TLR2, TLR4, TLR9, IL-4, IL-5 and IL-12, in addition to up-regulating TLR9, CD80 and CD86 expression. PS increased B-1b cell expression of TLR4, TLR9, CD40 and CD86, as well as IL-10 and IL-12 synthesis. Both of the treatments decreased the absolute number of B-1b cells in vitro, suggesting their early differentiation into B-1 cell-derived phagocytes (B-1CDP). We also observed a higher phagocytic activity from the phagocytes that were derived from B-1b cells after P. acnes and PS treatment. The adjuvant effect that P. acnes has on B-1 cells, mainly the B-1b subtype, reinforces the importance of B-1 cells in the innate and adaptive immune responses

Anatomical Basis of the Myofascial Trigger Points of the Gluteus Maximus Muscle

Myofascial pain syndrome is characterized by pain and limited range of motion in joints and caused by muscular contracture related to dysfunctional motor end plates and myofascial trigger points (MTrPs). We aimed to observe the anatomical correlation between the clinically described MTrPs and the entry point of the branches of the inferior gluteal nerve into the gluteus maximus muscle. We dissected twenty gluteus maximus muscles from 10 human adult cadavers (5 males and 5 females). We measured the muscles and compiled the distribution of the nerve branches into each of the quadrants of the muscle. Statistical analysis was performed by using Student’s t-test and Kruskal-Wallis tests. Although no difference was observed either for muscle measurements or for distribution of nerve branching among the subjects, the topography of MTrPs matched the anatomical location of the entry points into the muscle. Thus, anatomical substract of the MTrPs may be useful for a better understanding of the physiopathology of these disorders and provide basis for their surgical and clinical treatment

Differentiation of B-1b cells into B-1 cell-derived phagocytes (B-1CDP).

<p>The peritoneal cells from mice treated with <i>P. acnes</i>, PS or saline (control group) were cultured for 5 days. The non-adherent cells were re-cultured on cover glasses for 1 to 120 hours for phagocyte differentiation. The cells that adhered to the cover glasses were then fixed, stained with Giemsa and analyzed using optical microscopy. Magnification 500×.</p

Analysis of the activation status of B-1b lymphocytes <i>in vitro</i>.

<p>The non-adherent cell population that was obtained after 5 days in a B-1b enriched culture obtained from <i>P. acnes</i>-, PS-, or saline- (control group) treated mice was analyzed to determine TLR, co-stimulatory molecule, MHC II and cytokine expression in B-1b lymphocytes. The cells were stained with mAbs to identify the absolute numbers (A to C) of B-1b lymphocytes that expressed each molecule and the mean fluorescence intensity (MFI) of each marker (D to F). The absolute cell number and MFI are the means of two independent experiments with similar results. * <i>p</i><0.05 between the control and treated groups. ^#<i>p</i><0.05 between the <i>P. acnes</i> and PS treated groups.</p

Determination of the absolute number of B-1 cell subsets <i>in vivo</i> and <i>in vitro</i>.

<p>Peritoneal cells from <i>P. acnes</i>-, PS- or saline-treated mice were analyzed 24 h after treatment (<i>in vivo</i>). The non-adherent population of 5-day-old B-1b enriched cultures was also analyzed (<i>in vitro</i>). The cells were stained with mAbs to identify the B-1 cell subsets. (A to C) The small (R1) and large granular (R2) cells from the SSC-H×FSC-H dot plots are shown, followed by the CD23×CD19 analysis from R1 and R2 and the CD5×CD11b analysis from R3 and R4. (D and E) The absolute numbers of B-1b, B-1a and B-1c lymphocytes from mice treated with saline (control group), <i>P. acnes</i> and PS. The absolute numbers of the B-1a and B-1b lymphocytes are the sum of R1 and R2, and the B-1c values are derived from R1. The absolute cell number is the mean of two independent experiments with similar results. * <i>p</i><0.05 compared to the control group. ^#<i>p</i><0.05 between the <i>P. acnes</i> and PS treated groups.</p

Resveratrol Downmodulates Neutrophil Extracellular Trap (NET) Generation by Neutrophils in Patients with Severe COVID-19

The formation of microthrombi in lung autopsies indicates the involvement of NETs in the immunopathogenesis of severe COVID-19. Therefore, supplements inhibiting NET formation, in association with drugs with fewer adverse effects, should be a relevant strategy to attenuate the disease. Resveratrol (RESV) is a natural polyphenol with an important antiviral and antioxidant role. To modulate neutrophils from patients infected with SARS-CoV-2, we evaluated the in vitro effect of RESV on NET formation. Herein, we investigated 190 patients hospitalized with moderate, severe, and critical symptoms at Hospital das Clínicas, Brazil. We observed that neutrophilia in patients with severe COVID-19 infection is composed of neutrophils with activated profile able to release NET spontaneously. Notably, RESV decreased the neutrophil-activated status and the release of free DNA, inhibiting NET formation even under the specific PMA stimulus. At present, there is no evidence of the role of RESV in neutrophils from patients with COVID-19 infection. These findings suggest that adjunctive therapies with RESV may help decrease the inflammation of viral or bacterial infection, improving patient outcomes

Impact of Inflammatory Immune Dysfunction in Psoriasis Patients at Risk for COVID-19

Psoriasis is an immune-mediated dermatosis usually associated with comorbidities. Treatment varies from topicals to systemic drugs and data on susceptibility to viral infections in psoriatic patients are scarce. The objectives of this study were to analyze psoriatic patients on different therapies who were at risk for COVID-19 for seroprevalence of SARS-COV-2, pro-inflammatory cytokine profile, comorbidities and outcomes in order to unveil the immunological mechanisms involved in the anti-viral response in patients with psoriasis. Seventy-five patients with psoriasis were divided according to treatment: immunobiologics, methotrexate, topicals and acitretin. Twenty healthy controls were included. Plasma samples were collected for: IgG SARS-COV-2 (ELISA); IL-27, IL-29 and IL-18 (ELISA); and IL-1β, IL-17A, IL-6 and TNF (cytometric array). Seropositivity for SARS-COV-2 was detected in 24 out of 75 psoriasis patients and did not relate to COVID-19 symptoms and/or hospitalization, despite associated comorbidities. Psoriasis patients who were asymptomatic for SARS-COV-2 exhibited immune imbalance with high levels of IL-18, IL-17A and IL-6, and low levels of IL-27 compared to healthy controls. Psoriasis groups showed significant increased cytokine levels only in the group with immunobiologics. Despite immune deviations and lower IL-27, which has a potential antiviral impact, psoriatic patients did not exhibit complications related to COVID-19. An understanding of this kind of proinflammatory profile of psoriatic patients and of the lack of severe outcomes for COVID-19 is essential to establish novel therapeutic approaches and preventive measures, including with regard to the concomitance of viral infections