Duygu Sag

Background/aim: Kabasura kudineer choornam (KKC) is a polyherbal formulation of 15 ingredients. It has antiinflammatory and antimicrobial properties and are effective in managing the symptoms of H1N1 swine flu and COVID-19. However, its mechanism of action is not fully understood. In this study, we examined the effect of KKC on the polarization and function of primary human macrophages. Materials and methods: Human monocyte-derived macrophages (M0 macrophages) pretreated with KKC extract were polarized into M1, M2a, or M2c subtypes. The expression of the M1/M2 polarization markers was analyzed using qPCR, flow cytometry, and ELISA, and the phagocytosis capacity of macrophages was analyzed using flow cytometry. Results: Our data show that the KKC treatment increased the expression of the M1 markers IDO1, IL-1β, IL-12a (p35), and TNF in both polarized and unpolarized macrophages at mRNA level. However, it decreased the secretion of IL-12 (p70) in M1 macrophages and increased the secretion of TNF in M0, M2a, and M2c macrophages. IL-10 secretion was increased in M0 and M2a macrophages, while it was decreased in M1 macrophages after the KKC treatment. Interestingly, all KKC-treated macrophage phenotypes displayed a downregulation in the expression of the M1/M2 surface markers CD64, CD206, CD209, and CD163, which also play a role in phagocytosis. In accordance with this result, the phagocytic capacity of both polarized and unpolarized macrophages was decreased after the KKC treatment. Conclusion: KKC extract modulates macrophage inflammatory response and could be a potential supplement for the treatment of infectious and inflammatory diseases.

Background: Kabasura kudineer choornam ( KKC ) is a polyherbal formulation with 15 ingredients. It has been shown to have anti-inflammatory and anti-microbial properties and demonstrate efficacy in managing the symptoms of H1N1 swine flu and COVID-19. However, its mechanism of action is not fully comprehended. Purpose: Herein, we examined the effect of the KKC on the polarization and function of primary human macrophages. Methods: Human monocyte-derived macrophages (M0 macrophages) pre-treated with KKC extract were polarized into M1, M2a, or M2c subtypes. The expression of the M1/M2 polarization markers was analyzed by qPCR, flow cytometry, and ELISA, and the phagocytosis capacity of macrophages was analyzed by flow cytometry. Results: Our data show that the KKC treatment increased the expression of the M1 markers IDO1, IL-1β, IL-12a(p35), and TNF in both polarized and unpolarized macrophages at mRNA level. However, it decreased the secretion of IL-12 (p70) in M1 macrophages and increased the secretion of TNF in M0, M2a, and M2c macrophages. IL-10 secretion was increased in M0 and M2a macrophages, while it was decreased in M1 macrophages after the KKC treatment. Interestingly, all KKC -treated macrophage phenotypes displayed a downregulation in the expression of the M1/M2 surface markers CD64, CD206, CD209, and CD163, which also play a role in phagocytosis. In accordance with this result, the phagocytic capacity of both polarized and unpolarized macrophages was decreased after the KKC treatment. Conclusion: In conclusion, KKC extract modulates macrophage inflammatory response and could be a potential supplement for the treatment of infectious and inflammatory diseases.

Importance: Physicians are exposed to high stress and strain that results in burnout, which affects them, their families, their patients, and the entire health care system; thus, there is an urgent need to develop methods to increase the resiliency of physicians. Sudarshan Kriya Yoga (SKY) is a comprehensive yoga breathing and meditation-based program that is a potential approach to mitigate physician burnout.Objective: To determine whether SKY can reduce psychological distress and improve wellness in physicians.Design, Setting, and Participants: This randomized clinical trial assessed the potential efficacy of SKY compared with a stress management education (SME) training as control. This study was conducted online from November 11, 2021, to March 14, 2022, and included physicians from Turkey, Germany, and Dubai. Both the SKY and the SME control groups received 1.5 hours of training for 3 consecutive days via a group video conference call. Participants were physicians willing to do some form of relaxation exercise everyday for 2 months. Exclusion criteria included presence of major illness and maintaining a regular mind-body program practice. Statistical analysis took place from March to November 2023.Interventions: Participants were randomly assigned 1:1 into 2 groups—the SKY group or the SME (control) group—using a computer algorithm. After the 3-day instruction period, the participants in the SKY group practiced for approximately 30 minutes per day on their own and participated in a weekly 1-hour, group-based online follow-up practice. After the 3-day instruction period, participants in the SME group reviewed and applied the notes from stress management education training at their initiative and had a weekly 1-hour group-based online follow-up session.Main Outcomes and Measures: The primary outcomes were stress and depression (measured by the 42-item Depression, Anxiety, and Stress Scale [DASS-42]) and insomnia measured by the Regensburg Insomnia Scale (RIS) with primary end point at 8 weeks. Secondary outcomes included anxiety (DASS-42); optimism (Life Orientation Test-Revised [LOT-R]); professional fulfillment, work exhaustion, interpersonal disengagement, and overall burnout (Professional Fulfillment Index [PFI]); and self-reported professional errors (Self-Reported Professional Error Questionnaire).Results: This study included 129 participants (SME, 63 participants [48.9%]; SKY, 66 participants [51.1%]; 115 females [89.2%]; 14 males [10.8%]; mean [SD] age, 46.2 [9.0] years). Compared with the SME control group, participants in the SKY group had significantly decreased stress on the DASS-42 at posttraining (difference, −6.8 points; 95% CI, −9.6 to −4.1 points; P = .006) and at postintervention (difference, −6.0 points; 95% CI, −8.8 to −3.3 points; P = .03), significantly decreased depression at posttraining (difference, −5.7 points; 95% CI, −8.6 to −2.8 points; P < .001) and postintervention (difference, −5.4 points; 95% CI, −8.3 to −2.5 points; P < .001), and significantly decreased anxiety at postintervention. In addition, there was a significant decrease in insomnia from baseline to postintervention in the SKY group (difference, −0.3 points; 95% CI, −2.3 to 1.7 points; P = .01). The SKY group also showed significantly increased professional fulfillment as well as significant decreases in work exhaustion, interpersonal disengagement, and burnout. There was no effect on self-reported medical errors.Conclusions and Relevance: In this randomized clinical trial, physicians who regularly practiced SKY throughout a 2-month period experienced improvements in wellness and decreased burnout. These data suggest that SKY may be an effective, practical, and safe strategy to increase wellness and mitigate burnout in physicians.Trial Registration: ClinicalTrials.gov Identifier: NCT05956470

Background: TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily that can either induce cell death or activate survival pathways after binding to death receptors (DRs) DR4 or DR5. TRAIL is investigated as a therapeutic agent in clinical trials due to its selective toxicity to transformed cells. Macrophages can be polarized into pro-inflammatory/tumor-fighting M1 macrophages or anti-inflammatory/tumor-supportive M2 macrophages and an imbalance between M1 and M2 macrophages can promote diseases. Therefore, identifying modulators that regulate macrophage polarization is important to design effective macrophage-targeted immunotherapies. The impact of TRAIL on macrophage polarization is not known.Methods: Primary human monocyte-derived macrophages were pre-treated with either TRAIL or with DR4 or DR5-specific ligands and then polarized into M1, M2a, or M2c phenotypes in vitro. The expression of M1 and M2 markers in macrophage subtypes was analyzed by RNA sequencing, qPCR, ELISA, and flow cytometry. Furthermore, the cytotoxicity of the macrophages against U937 AML tumor targets was assessed by flow cytometry. TCGA datasets were also analyzed to correlate TRAIL with M1/M2 markers, and the overall survival of cancer patients.Results: TRAIL increased the expression of M1 markers at both mRNA and protein levels while decreasing the expression of M2 markers at the mRNA level in human macrophages. TRAIL also shifted M2 macrophages towards an M1 phenotype. Our data showed that both DR4 and DR5 death receptors play a role in macrophage polarization. Furthermore, TRAIL enhanced the cytotoxicity of macrophages against the AML cancer cells in vitro. Finally, TRAIL expression was positively correlated with increased expression of M1 markers in the tumors from ovarian and sarcoma cancer patients and longer overall survival in cases with high, but not low, tumor macrophage content.Conclusions: TRAIL promotes the polarization of human macrophages toward a proinflammatory M1 phenotype via both DR4 and DR5. Our study defines TRAIL as a new regulator of macrophage polarization and suggests that targeting DRs can enhance the anti-tumorigenic response of macrophages in the tumor microenvironment by increasing M1 polarization.

Background: TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily that can either induce cell death or activate survival pathways after binding to death receptors (DRs) DR4 or DR5. TRAIL is investigated as a therapeutic agent in clinical trials due to its selective toxicity to transformed cells. Macrophages can be polarized into pro-inflammatory/tumor-fighting M1 macrophages or anti-inflammatory/tumor-supportive M2 macrophages and an inbalance between M1 and M2 macrophages can promote diseases. Therefore, identifying modulators that regulate macrophage polarization is important to design effective macrophage-targeted immunotherapies. The impact of TRAIL on macrophage polarization is not known. Methods: Primary human monocyte-derived macrophages were pre-treated with either TRAIL or with DR4 or DR5-specific ligands and then polarized into M1, M2a, or M2c phenotypes in vitro . The expression of M1 and M2 markers in macrophage subtypes was analyzed by RNA sequencing, qPCR, ELISA, and flow cytometry. Furthermore, the cytotoxicity of the macrophages against U937 AML tumor targets was assessed by flow cytometry. TCGA datasets were also analyzed to correlate TRAIL with M1/M2 markers, and the overall survival of cancer patients. Results: TRAIL increased the expression of M1 markers at both mRNA and protein levels while decreasing the expression of M2 markers at the mRNA level in human macrophages. TRAIL also shifted M2 macrophages towards an M1 phenotype. Our data showed that both DR4 and DR5 death receptors play a role in macrophage polarization. Furthermore, TRAIL enhanced the cytotoxicity of macrophages against the AML cancer cells in vitro . Finally, TRAIL expression was positively correlated with increased expression of M1 markers in the tumors from ovarian and sarcoma cancer patients and longer overall survival in cases with high, but not low, tumor macrophage content. Conclusions: TRAIL promotes the polarization of human macrophages toward a proinflammatory M1 phenotype via both DR4 and DR5. Our study defines TRAIL as a new regulator of macrophage polarization and suggests that targeting DRs can enhance the anti-tumorigenic response of macrophages in the tumor microenvironment by increasing M1 polarization.

Astragaloside VII (AST VII), a triterpenic saponin isolated from Astragalus species, shows promise as a vaccine adjuvant, as it supported a balanced Th1/Th2 immune response in previous in vivo studies. However, the underlying mechanisms of its adjuvant activity have not been defined. Here, we investigated the impact of AST VII and its newly synthesized semi-synthetic analogs on human whole blood cells, as well as on mouse bone marrow-derived dendritic cells (BMDCs). Cells were stimulated with AST VII and its derivatives in the presence or absence of LPS or PMA/ionomycin and the secretion of cytokines and the expression of activation markers were analyzed using ELISA and flow cytometry, respectively. AST VII and its analogs increased the production of IL-1β in PMA/ionomycin-stimulated human whole blood cells. In LPS-treated mouse BMDCs, AST VII increased the production of IL-1β and IL-12, and the expression of MHC II, CD86, and CD80. In mixed leukocyte reaction, AST VII and derivatives increased the expression of the activation marker CD44 on mouse CD4⁺ and CD8⁺ T cells. In conclusion, AST VII and its derivatives strengthen pro-inflammatory responses and support dendritic cell maturation and T cell activation in vitro. Our results provide insights into the mechanisms of the adjuvant activities of AST VII and its analogs, which will be instrumental to improve their utility as a vaccine adjuvant.

Macrophages are highly plastic cells that can polarize into functionally distinct subsetsin vivoandin vitroin response to environmental signals. The development of protocols to model macrophage polarizationin vitrogreatly contributes to our understanding of macrophage biology. Macrophages are divided into two main groups: Pro-inflammatory M1 macrophages (classically activated) and anti-inflammatory M2 macrophages (alternatively activated), based on several key surface markers and the production of inflammatory mediators. However, the expression of these common macrophage polarization markers is greatly affected by the stimulation time used. Unfortunately, there is no consensus yet regarding the optimal stimulation times for particular macrophage polarization markers inin vitroexperiments. This situation is problematic, (i) as analysing a particular marker at a suboptimal time point can lead to false-negative results, and (ii) as it clearly impedes the comparison of different studies. Using human monocyte-derived macrophages (MDMs)in vitro, we analysed how the expression of the main polarization markers for M1 (CD64, CD86, CXCL9, CXCL10, HLA-DR, IDO1, IL1β, IL12, TNF), M2a (CD200R, CD206, CCL17, CCL22, IL-10, TGM2), and M2c (CD163, IL-10, TGFβ) macrophages changes over time at mRNA and protein levels. Our data establish the most appropriate stimulation time for the analysis of the expression of human macrophage polarization markersin vitro. Providing such a reference guide will likely facilitate the investigation of macrophage polarization and its reproducibility.

Various aspects of the in vitro culture conditions can impact the functional response of immune cells. For example, it was shown that a Ca ²⁺ concentration of at least 1.5 mM during in vitro stimulation is needed for optimal cytokine production by conventional αβ T cells. Here we extend these findings by showing that also unconventional T cells (invariant Natural Killer T cells, mucosal-associated invariant T cells, γδ T cells), as well as B cells, show an increased cytokine response following in vitro stimulation in the presence of elevated Ca ²⁺ concentrations (approx. 1.8 mM). This effect appeared more pronounced with mouse than with human lymphoid cells and did not influence their survival. A similarly increased cytokine response due to elevated Ca ²⁺ levels was observed with primary human monocytes. In contrast, primary human monocyte-derived macrophages, either unpolarized (M0) or polarized into M1 or M2 macrophages, displayed increased cell death in the presence of elevated Ca ²⁺ concentrations. Furthermore, elevated Ca ²⁺ concentrations promoted phenotypic M1 differentiation by increasing M1 markers on M1 and M2 macrophages and decreasing M2 markers on M2 macrophages. However, the cytokine production of macrophages, again in contrast to the lymphoid cells, was unaltered by the Ca ²⁺ concentration. In summary, our data demonstrate that the Ca ²⁺ concentration during in vitro cultures is an important variable to be considered for functional experiments and that elevated Ca ²⁺ levels can boost cytokine production by both mouse and human lymphoid cells.

Astragaloside VII (AST VII), a plant triterpenoid saponin isolated from Astragalus species, shows promise as vaccine adjuvant, as it supports a balanced Th1/Th2 immune response. However, the underlying mechanisms of its adjuvant activity have not been defined. Here we investigated the impact of AST VII and its newly synthesized semi-synthetic analogs on human whole blood cells, as well as on mouse bone marrow-derived dendritic cells (BMDCs). Cells were stimulated with AST VII and its derivatives in the presence or absence of LPS or PMA/ionomycin and the secretion of cytokines and the expression of activation markers were analyzed by ELISA and flow cytometry, respectively. AST VII and its analogs increased the production of IL-1β in PMA/ionomycin stimulated human whole blood cells. In LPS-treated mouse BMDCs, AST VII increased the production of IL-1β and IL-12, and the expression of MHC II, CD86, and CD80. The strength of the IL-1β boost correlated directly with the hydrophobicity of the AST VII compounds. In mixed leukocyte reaction, AST VII and derivatives increased the expression of the activation marker CD44 on mouse CD4 ⁺ and CD8 ⁺ T cells. In conclusion, AST VII and its derivatives strengthen pro-inflammatory responses, support dendritic cell maturation, and T cell activation in vitro . Our results provide insights into the mechanisms of the adjuvant activities of AST VII and its analogs, which will be instrumental to improve their utility as vaccine adjuvant.

Macrophages are highly plastic cells that can polarize into functionally distinct subsets in vivo and in vitro in response to environmental signals. The development of protocols to model macrophage polarization in vitro greatly contributes to our understanding of macrophage biology. Macrophages are divided into two main groups: Pro-inflammatory M1 macrophages (classically activated) and anti-inflammatory M2 macrophages (alternatively activated), based on several key surface markers and the production of inflammatory mediators. However, the expression of these common macrophage polarization markers is greatly affected by the stimulation time used. Unfortunately, there is no consensus yet regarding the optimal stimulation times for particular macrophage polarization markers in in vitro experiments. This situation is problematic, (i) as analysing a particular marker at a suboptimal time point can lead to false-negative results, and (ii) as it clearly impedes the comparison of different studies. Using human monocyte-derived macrophages (MDMs) in vitro , we analysed how the expression of the main polarization markers for M1 (CD64, CD86, CXCL9, CXCL10, HLA-DR, IDO1, IL1β, IL12, TNF), M2a (CD200R, CD206, CCL17, CCL22, IL-10, TGM2), and M2c (CD163, IL-10, TGFβ) macrophages changes over time at mRNA and protein levels. Our data establish the most appropriate stimulation time for the analysis of the expression of human macrophage polarization markers in vitro . Providing such a reference guide will likely facilitate the investigation of macrophage polarization and its reproducibility.