Number Of CD11b Research Articles

The Protective Effect of Astragalus Polysaccharide on Experimental Autoimmune Encephalomyelitis in Mice by Activating the AMPK/JAK/STAT3/Arginase-1 Signaling Pathway.

This study aimed to investigate the protective effect and mechanism of Astragalus polysaccharide (APS) on autoimmune encephalomyelitis. C57BL/6 mice were randomly divided into the blank control group, EAE group, and APS intervention group (n=15/group). The Experimental Autoimmune Encephalomyelitis (EAE) mouse model was established by active immunization. The pathological changes in the spinal cord were evaluated by Hematoxylin-eosin (HE) and Luxol Fast Blue (LFB) staining. The number of CD11b+ Gr-1+ myeloid-derived suppressor cells (MDSCs) in the spleen tissues of mice in each group was determined by immunofluorescence staining. The expression of Arginase-1 in the spinal cord and spleen of each group was detected by immunofluorescence double staining. The TNF-α, IL-6, and Arginase-1 levels in the spleen were detected by ELISA assay. A western blot was used to detect the protein expression of the AMPK/JAK/STAT3/Arginase-1 signaling pathway. After the intervention of APS, the incidence of autoimmune encephalomyelitis in mice of the APS group was significantly lower than that in the EAE group, and the intervention of APS could significantly delay the onset time in the EAE mice, and the score of neurological function deficit in mice was significantly lower than that in EAE group (P < 0.05). APS intervention could reduce myelin loss and improve the inflammatory response of EAE mice. Moreover, it could induce the expression of CD11b+ GR-1 + bone MDSCs in the spleen and increase the expression of Arginase-1 in the spinal cord and spleen. This study further demonstrated that APS can protect EAE mice by activating the AMPK/JAK/STAT3/Arginase-1 signaling pathway. After the intervention of APS, myelin loss and inflammatory response of EAE mice were effectively controlled. APS promoted the secretion of Arginase-1 by activating MDSCs and inhibited CD4+T cells by activating AMPK/JAK/STAT3/Arginase-1 signaling pathway, thus improving the clinical symptoms and disease progression of EAE mice.

CD11b+ миелоидные клетки селезенки при карциномах

Introduction. Splenic myeloid cells include monocytes and myeloid suppressor cells, which are deposited in the spleen and recruited to the sites of chronic inflammation, reparative tissues, and tumor microenvironment. In vivo experiments are mainly used to study the influence of the splenic system on the development and progression of the malignant neoplasm. The characteristics of myeloid elements with a common marker (CD11b) that are located in different splenic morphological and functional zones and their relation to metastases are still poorly studied. We aimed to compare the number and phenotypic features of CD11b+ myeloid cells considering their location in different splenic structural and functional areas in cohorts of patients with and without a malignant neoplasm, as well as depending on hematogenous and lymphogenous metastases in patients with carcinomas of various locations. Materials and methods. To estimate the number of CD11b+ cells with different variants of CD45, CD34, and CD90 expression, we applied the TSA technique. The number of cells of each phenotype per 1 mm2 of follicular, marginal, and red pulp areas was calculated in both groups. The association of cell number with lymphogenic and distant metastases was investigated in the group of patients with malignant neoplasms. Results. The CD11b+ myeloid cell population is heterogeneous and represented by cells at different stages of monocyte/MDSC differentiation, whose numbers predominate in the red pulp. The following changes are associated with carcinomas: a 17-fold increase in the number of promonocytes in the red pulp, a 2-fold decrease in the number of myelopoiesis progenitors in the marginal zone, and a reduction in the quantity of immature monocytoid cells/monoblasts in the marginal zone and the red pulp (5- and 12.5-fold, respectively). The number of CD11b+ myeloid cells with such phenotypes decreased even more in the cases with lymphogenic metastases. No association was found with distant metastases. Conclusion. The number of CD11b+ splenic myeloid cells is associated with carcinomas and lymphogenic metastases. Keywords: spleen, myeloid cells, CD11b, carcinomas, metastasis

Spleen contributes to chronic restraint stress-induced lung injury through splenic CD11b+ cells

Chronic stress can induce lung injury. The spleen, as the largest peripheral immune organ, plays a crucial role in various lung diseases. Our previous study found that the spleen underwent significant changes during chronic restraint stress (CRS). However, the exact role of the spleen in CRS-induced lung injury remains unclear. In this study, we found that CRS could increase lung index. CRS could lead to alterations of the lungs such as destruction of alveolar wall, thickening of alveolar septa, dilation of pulmonary capillaries, and increased inflammatory cell infiltration. CRS increases the concentration of malondialdehyde (MDA), decreases the level of surfactant protein A (SP-A), and elevates the levels of pro-inflammatory factors (TNF-α, IL-6, and IL-1β) in the lungs. Additionally, CRS could increase the proportions and numbers of CD11b+Ly6ChiLy6G- monocytes in the lung, while cannot alter proportions and numbers of CD3-NK1.1+ NK cells, CD3+CD4+ T cells, CD3+CD8+ T cells, and CD11b+Ly6G+ neutrophils. Moreover, the levels of inflammatory markers in lung tissues were positively correlated with the proportion of CD11b+Ly6ChiLy6G- monocytes. Interestingly, splenectomy inhibited CRS-induced lung injury and attenuated the alteration in the proportion of CD11b+Ly6ChiLy6G- monocytes in the lungs induced by CRS. Moreover, splenic CD11b+ cells, rather than splenic CD11b- cells, transfused into splenectomized mice, and subsequently exposed to CRS, can cause lung injury. These results suggest that CRS could induce lung injury and CD11b+Ly6ChiLy6G- monocytes aggregation in the lung. The spleen could contribute to CRS-induced lung injury. Furthermore, splenic CD11b+ cells might play an important role in CRS-induced lung injury.

Clinical and immunological efficacy of immunomodulating hexapeptide associated with the restoration of CD11b&lt;sup&gt;+&lt;/sup&gt;CD64&lt;sup&gt;-&lt;/sup&gt;CD32&lt;sup&gt;+&lt;/sup&gt;CD16&lt;sup&gt;+&lt;/sup&gt; and CD11b&lt;sup&gt;+&lt;/sup&gt;CD64&lt;sup&gt;+&lt;/sup&gt;CD32&lt;sup&gt;+&lt;/sup&gt;CD16&lt;sup&gt;+&lt;/sup&gt; neutrophil granulocytes subset in women with chronic infectious and inflammatory diseases of the pelvic organs

Failure of anti-infectious immune protection is considered a reason for the prolonged course and recurrence of chronic infectious and inflammatory diseases of pelvic organs (PID). Our aim was to evaluate the effect of an original hexapeptide (HP) on negatively altered subpopulations of neutrophil granulocytes (NG) CD11b+CD64-CD32+CD16+ and CD11b+CD64+CD32+CD16+, their phenotype and associated effector functions in immunocompromised women with PID.&#x0D; 35 women (20-40 years old) with PID were studied during the period of clinical exacerbation (study group 1, SG1). Study group 1a (SG1a) consisted of patients who underwent treatment including the HP injections (45 mcg/ mL, 1 ml intramuscularly once a day for 10 days). The comparison group (CG) consisted of 20 conditionally healthy women. The numbers of CD11b+CD64-CD32+CD16+NG and CD11b+CD64+CD32+CD16+NG cell subsets and the density of receptor expression, phagocytic and microbicidal function of NG were determined.&#x0D; In SG1, decreased counts of the major NG subpopulation (CD11b+CD64-CD32+CD16+NG) was revealed (p 0.05), with a trend for increase of minor subset CD11b+CD64+CD32+CD16+NG (p 0.05). In the CD11b+CD64-CD32+CD16+NG subset, we noted a decreased expression of CD16 (1.4-fold), CD11b (2-fold) (p1, 2 0.05). In the minor subset CD11b+CD64+CD32+CD16+NG, the expression densities were decreased in CD16 (1.7-fold), CD11b (2.1-fold, p1, 2 0.05). At the same time, the phagocytic and microbicidal functions of NG were found to be decreased. In the course of immunomodulatory therapy with the HP-based drug, positive changes in immunological parameters were revealed. In SG1a, an increased number of major NC subset was observed, with an increase in the expression density of CD16 by 1.2 times, CD11b by 1.7 times relative to SG1 (p1, 2 0.05). The contents of minor NG subset tended to decrease, along with CD16 expression density reaching the indices of comparison group. CD11b increased 1.3 times relative to SG1 (p 0.05). Higher ratios of actively phagocytizing NG and their killing ability have been registered. Clinically, we observed faster regression of clinical PID exacerbation symptoms and decreased frequency of relapses 6 months after treatment in 88.6% of cases. The positive immunomodulatory effects of the HP-based drug upon altered subsets of CD11b+CD64-CD32+CD16+ and CD11b+CD64+CD32+CD16+ NGs, their phenotype and associated effector functions suggest an opportunity of its usage for the correction of NG dysfunctions in immunocompromised women with PID, thus providing stable clinical and immunological remission and protective effect.

Lentinula Edodes Mycelia extract regulates the function of antigen-presenting cells to activate immune cells and prevent tumor-induced deterioration of immune function

Immune cell activation is essential for cancer rejection; however, the tumor microenvironment leads to deterioration of immune function, which enables cancer cells to survive and proliferate. We previously reported that oral ingestion of Lentinula Edodes Mycelia (L.E.M.) extract enhances the tumor antigen-specific T-cell response and exerts an antitumor effect in a tumor-bearing mouse model. In this study, we focused on antigen-presenting cells (APCs) located upstream of the immune system, induced a T-cell response, then examined the impact of L.E.M. extract on the APCs. L.E.M. extract enhanced the expression of MHC-I, MHC-II, CD86, CD80, and CD40 in bone marrow-derived dendritic cells (DCs) and strongly induced the production of IL-12. L.E.M.-stimulated DCs enhanced IFN-γ production from CD8+ T cells and induced their differentiation into effector cells. Furthermore, L.E.M. extract promoted IL-12 production and suppressed the production of IL-10 and TGF-β by transforming bone marrow-derived macrophages into M1-like macrophages. Furthermore, in a B16F10 melanoma inoculation model, DCs in the spleen were decreased and their activation was suppressed by the presence of cancer; however, ingestion of L.E.M. extract prevented this functional deterioration of DCs. In the spleen of cancer-bearing mice, the number of CD11b− F4/80+ macrophages in a hypoactivated state was also increased, whereas L.E.M. extract suppressed the increase of such macrophages. These findings suggest that L.E.M. extract may exhibit an antitumor immune response by regulating the function of APCs to induce cytotoxic T lymphocytes, as well as by suppressing the decline in antigen-presenting cell activity caused by the presence of cancer.

1487-P: Inosine Monophosphate Preferentially Regulates Myeloid Progenitor Differentiation toward Neutrophil Production in Diabetes

Previous studies described hyperglycemia-induced myelopoiesis and atherosclerotic progression in mice with type I diabetes. By targeted metabolomics, we reported the increase of inosine monophosphate (IMP) in granulocyte/monocyte progenitors (GMP) in db/db mice. Moreover, IMP treatment stimulated GMP proliferation, resulting in skewed myelopoiesis and enhanced acute pancreatitis progression. However, how IMP modulates granulocyte production is not known. To investigate that, equal amount of FACS-sorted GMP of wild-type mice were cultivated with PBS or 15 mM IMP for 5 days in vitro. FACS analysis revealed that the absolute number of neutrophils was 1.7-fold higher but the number of CD11b+ monocytes was 3.2-fold lower in IMP group compared to control (p&amp;lt;0.05 for both). Thereafter, FACS-sorted GMP treated with or without IMP for 3 days were harvested for single cell RNA sequencing. When classified by marker gene expression, 19 clusters representing 7 cell types were identified including neutrophils, monocytes, GMP, intermediate GMP/neutrophils, basophils, Megakaryocyte erythroid progenitors/megakaryocytes (MEP/Mgk), and platelets. The proportions of neutrophils and MEP/Mgk increased 1.3-, and 1.6-fold in IMP-treated GMP cells compared with controls (p=0.059 for neutrophils; p=0.032 for MEP/Mgk). Nevertheless, the percentage of other cell populations were comparable between two groups (p≥0.08 for all). Furthermore, cycling neutrophils in cluster 3 was 1.4-fold increase in IMP-treated GMP cells compared with controls (p=0.04). Accordingly, GO analysis and Gene set enrichment analysis illustrated that genes involved in cell cycles was enriched in cycling neutrophil clusters. In conclusion, our data demonstrated that IMP preferentially regulated GMP proliferation and differentiation toward neutrophil production, which provides new insights on the crosstalk between skewed metabolites and inflammation in diabetes. Disclosure Y.Dong: None. C.Yan: None. Y.Zhang: None. X.Yang: None. Y.Feng: None. Funding National Natural Science Foundation of China (81670765, 82070841)

Effects and mechanism of annexin A1-overexpressing human adipose-derived mesenchymal stem cells in the treatment of mice with acute respiratory distress syndrome

Effects and mechanism of annexin A1-overexpressing human adipose-derived mesenchymal stem cells in the treatment of mice with acute respiratory distress syndrome

Intestinal colonization with Candida auris and mucosal immune response in mice treated with cefoperazone oral antibiotic.

Candida auris, an emerging multi-drug resistant fungal pathogen, causes invasive infections in humans. The factors regulating the colonization of C. auris in host niches are not well understood. In this study, we examined the effect of antibiotic-induced gut dysbiosis on C. auris intestinal colonization, dissemination, microbiome composition and the mucosal immune response. Our results indicate that mice treated with cefoperazone alone had a significant increase in C. auris intestinal colonization compared to untreated control groups. A significant increase in the dissemination of C. auris from the intestine to internal organs was observed in antibiotic-treated immunosuppressed mice. Intestinal colonization of C. auris alters the microbiome composition of antibiotic-treated mice. Relative abundance of firmicutes members mainly Clostridiales and Paenibacillus were considerably increased in the cefoperazone-treated mice infected with C. auris compared to cefoperazone-treated uninfected mice. Next, we examined the mucosal immune response of C. auris infected mice and compared the results with Candida albicans infection. The number of CD11b+ CX3CR1+ macrophages was significantly decreased in the intestine of C. auris infected mice when compared to C. albicans infection. On the other hand, both C. auris and C. albicans infected mice had a comparable increase of the number of Th17 and Th22 cells in the intestine. A significant increase in Candida-specific IgA was observed in the serum of C. auris but not in the C. albicans infected mice. Taken together, treatment with broad-spectrum antibiotic increased the colonization and dissemination of C. auris from the intestine. Furthermore, findings from this study for the first time revealed the microbiome composition, innate and adaptive cellular immune response to intestinal infection with C. auris.

Assessment of the immune status of women after ablative fractional laser photothermolysis procedure for the correct of involutional facial skin changes

Introduction. Ablative fractional laser photothermolysis (A-FLPh), used for rejuvenation of aging skin, is based on its controlled damage. Factors of the immune system are involved in the reparative regeneration processes triggered, which, in turn, is also subject to age-related remodeling or immunostaining. The aim of the work was to evaluate in dynamics the response of the immune system to the A-FLPh procedure performed for correction of age-associated facial skin changes. Materials and methods. The study included 25 women aged 42 to 55 years who underwent A-FLPh treatment of facial skin with an Erbium laser. The number of leukocytes, monocytes, neutrophils, lymphocytes, T-lymphocytes, T-helpers, cytotoxic T-cells, regulatory T-cells, NKT-lymphocytes, NK-lymphocytes were counted in the peripheral blood before, on the 8th and 24th after the procedure. We studied phagocytic function of neutrophils and monocytes, NBT-reducing and lysosomal activity of neutrophils; determined the amount of IgA, IgM, IgG, IL-4, IL-6, IL-8, IL-10, circulating immune complexes (CIC). Results. On the 8th day after A-FLPh, the number of neutrophils, neutrophils and monocytes phagocytosis, the number of lymphocytes, regulatory T-cells, IL-6 and IL-8 levels significantly increased; in parallel, the number of CD11b+ NK-lymphocytes, CD11b+ NKT-lymphocytes, IgA, IgG, IL-10 concentrations decreased. On the 24th day, quantitative functional indices of neutrophils, total number of lymphocytes, concentrations of IgA and IgG had no reliable difference from pre-procedure values, phagocytic parameters of monocytes, number of regulatory T-cells, IL-6 and IL-8 levels remained significantly higher, while the number of CD11b+ NK-lymphocytes, CD11b+ NKT-lymphocytes and IL-10, on the contrary, significantly lower than the initial level. Discussion. The revealed changes of systemic immunity indices after A-FLPh testify to both direct and regulatory-modulatory influence of immune factors on skin repair and remodeling after laser damage. Conclusion. The A-FLPh procedure induces a response from both cellular and humoral factors of the immune system, predominantly innate immunity.

Abstract P339: A Possible Role Of Neuronal Adam17 In The Development Of Hypertension And Related Cognitive Impairment By Altering The Microenvironment

Hypertension is one of the most prevalent cardiovascular diseases worldwide and is known to be dominated by the sympathetic nervous system (SNS). Previously, we found that targeting A Disintegrin and Metalloproteinase 17 (ADAM17) in glutamatergic neurons was able to blunt Ang-II-induced excitation of pre-autonomic neurons, and blocked DOCA-salt treatment-induced sympatho-excitation in mice, thereby alleviating their development of hypertension. However, how ADAM17 support the activation of pre-autonomic neurons remains unknown. Beyond the glutamatergic neurons, we found in the current study that microglial activation and increase of pro-inflammatory cytokine levels in the hypothalamus, induced by DOCA-salt treatment, were blunted in those mice with ADAM17 knockout in glutamatergic neurons (A17G; number of CD11b+cells per line: NT+DOCA vs. A17G+DOCA= 11±2 vs. 6±1, P= 0.0017; normalized TNFα level: NT+DOCA vs. A17G+DOCA= 1.00±0.37 vs. 0.67±0.11, P&lt; 0.05; normalized IL-6 level: 1.00±0.32 vs. 0.59±0.20, P&lt; 0.05). Especially, it also reversed DOCA-salt-induced downregulation of Gad67 mRNA expression (NT= 1.00±0.11, NT+DOCA= 0.66±0.12, A17G= 1.02±0.15, A17G+DOCA= 1.67±0.24 fold of change, NT vs. NT+DOCA and A17G vs. A17G+DOCA: P&lt; 0.05), and normalized the decreased level of GABA in hypothalamus (normalized GABA levels: NT= 1.00±0.15, NT+DOCA= 0.84±0.09, A17G+DOCA= 1.09±0.17, NT vs. NT+DOCA: P&lt; 0.05), suggesting that in addition to the effect of ADAM17 on the neuron itself, it might also regulate the SNS through altering the neuronal microenvironment. In L-NAME hypertensive model, though A17G mice showed no improvement in NOS-blockade-induced dysfunction of neuro-vascular coupling (increase of CBF induced by whisker-stimulation: NT vs. A17G= 24.34±11.20 vs. 24.87±15.78 PU%), it did exhibit improved performance in Barnes maze test (latency time: NT vs. A17G= 32.77±19.36 vs. 95.54±59.03 s, P= 0.002), suggesting that ADAM17 in glutamatergic is critical for the process of neuronal damage induced by hypoxia and ischemia, possibly through modulating the homeostasis of neuronal microenvironment.

Effects of splenectomy on skin inflammation and psoriasis-like phenotype of imiquimod-treated mice

Imiquimod (IMQ) is widely used as animal model of psoriasis, a chronic inflammatory skin disorder. Although topical application of IMQ to back skin causes splenomegaly in mice, how the spleen affects the psoriasis-like phenotype of IMQ-treated mice remains unclear. In this study, we analyzed the cellular composition of spleen and measured metabolites in blood of IMQ-treated mice. We also investigated whether splenectomy influences the degree of skin inflammation and pathology in IMQ-treated mice. Flow cytometry showed that the numbers of CD11b+Ly6c+ neutrophils, Ter119+ proerythroblasts, B220+ B cells, F4/80+ macrophages, and CD11c+ dendritic cells in the spleen were significantly higher in IMQ-treated mice compared to control mice. An untargeted metabolomics analysis of blood identified 14 metabolites, including taurine and 2,6-dihydroxybenzoic acid, whose levels distinguished the two groups. The composition of cells in the spleen and blood metabolites positively correlated with the weight of the spleen. However, splenectomy did not affect IMQ-induced psoriasis-like phenotypes compared with sham-operated mice, although splenectomy increased the expression of interleukin-17A mRNA in the skin of IMQ-treated mice. These data suggest that the spleen does not play a direct role in the development of psoriasis-like phenotype on skin of IMQ-treated mice, though IMQ causes splenomegaly.

Nonerythropoietic Erythropoietin Mimetic Peptide ARA290 Ameliorates Chronic Stress-Induced Depression-Like Behavior and Inflammation in Mice

Major depressive disorder (MDD) is a highly prevalent psychiatric disorder. But the treatment of depression remains challenging. Anti-inflammatory treatments frequently produce antidepressant effects. EPO-derived helix-B peptide ARA290 has been reported to retain the anti-inflammatory and tissue-protective functions of EPO without erythropoiesis-stimulating effects. The effects of ARA290 on MDD remain elusive. This study established chronic unpredictable mild stress and chronic social defeat stress mouse models. Daily administration of ARA290 during chronic stress induction in two mouse models ameliorated depression-like behavior, similar to fluoxetine. With marginal effects on peripheral blood hemoglobin and red cells, ARA290 and fluoxetine reversed chronic stress-induced increased frequencies and/or numbers of CD11b+Ly6Ghi neutrophils and CD11b+Ly6Chi monocytes in the bone marrow and meninges. Furthermore, both drugs reversed chronic stress-induced microglia activation. Thus, ARA290 ameliorated chronic stress-induced depression-like behavior in mice through, at least partially, its anti-inflammatory effects.

Abstract 2191: Alteration of anti-tumoral immunity in the pre-metastatic bone microenvironment via autonomic nerve system dysfunction

Abstract Bone metastasis is a major cause of morbidity and mortality for breast cancer patients. Bone is a unique metastatic microenvironment because of complex interactions among numerous distinct cell types such as osteo-blasts, -clasts, -cytes and marrow immune cells in physiological and pathological conditions. Imbalanced bone homeostasis by diverse factors such as the sympathetic nerve system (SNS) activation, potentially contribute to the progression of bone metastasis, yet precise mechanisms remain unclear. We investigated the effects of beta 2 adrenergic receptor (β2AR) activation in osteoblasts induced by prolonged SNS stimulation on anti-tumoral immunity in bone metastasis. We treated female Balb/C with chronic immobilization stress (CIS; 2 h. daily) and found that CIS increased syngeneic intra-tibial 4T1 bone metastasis growth as well as the number of CD11b+Gr1+ myeloid-derived suppressor cells (MDSC) counterbalancing anti-tumoral cytotoxic T cells in bone microenvironment. A β2AR-specific inhibitor (ICI-118551) or MDSC depletion using anti-Gr1 antibodies reversed CIS-induced bone metastatic tumor growth. More importantly, 2-week CIS pre-treatment increased EDU+ MDSC proliferation in the bone marrow, contributing to subsequent bone metastatic tumor growth and micro-metastatic seeding in intra-tibial and intra-cardiac tumor injection models, respectively. When CIS was administered after the establishment of bone metastatic tumors, the pro-tumorigenic effects were marginal. In osteoblast-specific β2AR knockout mice (Adrb2flox/flox; murine 2.3kb Col1-CreC57BL6 mice), CIS did not increase MDSC proliferation compared with littermate floxed controls, suggesting that β2AR in osteoblasts upregulates MDSC in bone microenvironment. RNA-sequencing transcriptome analysis of saline- or clenbuterol (a selective β2AR agonist)-treated murine osteoblasts showed that interleukin-6 (IL6) was the most significantly increased cytokine. Indeed, CIS or clenbuterol treatment increased IL-6 expression in vivo (immunohistochemistry) and in vitro (cultured osteoblasts), respectively. MDSC isolated from the bone marrow of CIS-treated mice had significantly increased STAT3 phosphorylation by flow cytometry and the expression of immuno-suppressive functional molecules such as arginase 1 (Arg1) and indolamine 2,3-dioxygenase 1 (Ido1) compared to those from control mice. Our data collectively demonstrated that SNS-dependent β2AR in osteoblasts expands and activates MDSC in the pre-metastatic bone microenvironment via the IL6-STAT3-Arg1/IDO1 pathway, creating a fertile soil for bone metastasis progression. Our data provided the first direct evidence for anti-tumoral immunity and pro-tumorigenic effects of autonomous nerve system dysfunction specific to the metastatic bone microenvironment. Citation Format: Eun Jung Lee, Da Hyeon Yun, Seungpil Jung, Serk In Park. Alteration of anti-tumoral immunity in the pre-metastatic bone microenvironment via autonomic nerve system dysfunction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2191.

Rejuvenation of neutrophils and their extracellular vesicles is associated with enhanced aged fracture healing.

Tissue repair is negatively affected by advanced age. Recent evidence indicates that hematopoietic cell‐derived extracellular vesicles (EVs) are modulators of regenerative capacity. Here, we report that plasma EVs carrying specific surface markers indicate the degree of age‐associated immunosenescence; moreover, this immunosenescence phenotype was accentuated by fracture injury. The number of CD11b+Ly6CintermediateLy6Ghigh neutrophils significantly decreased with age in association with defective tissue regeneration. In response to fracture injury, the frequencies of neutrophils and associated plasma EVs were significantly higher in fracture calluses than in peripheral blood. Exposure of aged mice to youthful circulation through heterochronic parabiosis increased the number of neutrophils and their correlated Ly6G+ plasma EVs, which were associated with improved fracture healing in aged mice of heterochronic parabiosis pairs. Our findings create a foundation for utilizing specific immune cells and EV subsets as potential biomarkers and therapeutic strategies to promote resilience to stressors during aging.

Carabin Deficiency Aggravates Hepatic Ischemia-Reperfusion Injury Through Promoting Neutrophil Trafficking via Ras and Calcineurin Signaling.

Neutrophil infiltration plays an important role in the initial phase of hepatic ischemia and reperfusion injury (HIRI). Despite many different key molecules that have been reported to meditate neutrophil trafficking in HIRI, the mechanism of this process has not been fully elucidated. In this study, we found that Carabin deficiency in myeloid cells (LysMCre : Carabinfl/fl) aggravated IRI-induced hepatic injury and apoptosis through increasing the infiltration of CD11b+Ly6G+ neutrophils. ImmGen Datasets further revealed that Carabin was expressed in bone marrow neutrophils (GM.BM) but was significantly downregulated in thio-induced peripheral neutrophils (GN.Thio.PC), which was consistently verified by comparing GM.BM and liver-infiltrating neutrophils induced by IRI. Mechanistically, up-regulation of Carabin in GM.BM in vitro reduced the expression levels of P-selectin, E-selectin, and αvβ3 integrin through inhibiting Ras-ERK and Calcineurin-NFAT signaling. Furthermore, blocking P-selectin, E-selectin, and αvβ3 integrin in LysMCre : Carabinfl/fl mice decreased the frequency and number of CD11b+Ly6G+ neutrophils and reversed hepatic ischemia−reperfusion damage. In conclusion, our results provide a new understanding of Carabin, such that it is expressed and functions not only in adaptive immune cells (T and B cells) but also in innate immune cells (neutrophils), contributing to the migration of neutrophils. These findings provide novel and promising therapeutic targets for the prevention of HIRI during liver transplantation or hepatic surgery.

Early-phase administration of human amnion-derived stem cells ameliorates neurobehavioral deficits of intracerebral hemorrhage by suppressing local inflammation and apoptosis

BackgroundIntracerebral hemorrhage (ICH) is a significant cause of death and disabilities. Recently, cell therapies using mesenchymal stem cells have been shown to improve ICH-induced neurobehavioral deficits. Based on these findings, we designed this study to evaluate the therapeutic efficacy and underlying mechanisms by which human amnion-derived stem cells (hAMSCs) would ameliorate neurobehavioral deficits of ICH-bearing hosts.MethodshAMSCs were induced from amnia obtained by cesarean section and administered intravenously to ICH-bearing mice during the acute phase. The mice were then subject to multitask neurobehavioral tests at the subacute phase. We attempted to optimize the dosage and timing of the hAMSC administrations. In parallel with the hAMSCs, a tenfold higher dose of human adipose-derived stem cells (ADSCs) were used as an experimental control. Specimens were obtained from the ICH lesions to conduct immunostaining, flow cytometry, and Western blotting to elucidate the underlying mechanisms of the hAMSC treatment.ResultsThe intravenous administration of hAMSCs to the ICH-bearing mice effectively improved their neurobehavioral deficits, particularly when the treatment was initiated at Day 1 after the ICH induction. Of note, the hAMSCs promoted clinical efficacy equivalent to or better than that of hADSCs at 1/10 the cell number. The systemically administered hAMSCs were found in the ICH lesions along with the local accumulation of macrophages/microglia. In detail, the hAMSC treatment decreased the number of CD11b+CD45+ and Ly6G+ cells in the ICH lesions, while splenocytes were not affected. Moreover, the hAMSC treatment decreased the number of apoptotic cells in the ICH lesions. These results were associated with suppression of the protein expression levels of macrophage-related factors iNOS and TNFα.ConclusionsIntravenous hAMSC administration during the acute phase would improve ICH-induced neurobehavioral disorders. The underlying mechanism was suggested to be the suppression of subacute inflammation and apoptosis by suppressing macrophage/microglia cell numbers and macrophage functions (such as TNFα and iNOS). From a clinical point of view, hAMSC-based treatment may be a novel strategy for the treatment of ICH.

Endosomal Sequestration of TLR4 Antibody Induces Myeloid-Derived Suppressor Cells and Reverses Acute Type 1 Diabetes.

We previously showed that treating NOD mice with an agonistic monoclonal anti-TLR4/MD2 antibody (TLR4-Ab) reversed acute type 1 diabetes (T1D). Here, we show that TLR4-Ab reverses T1D by induction of myeloid-derived suppressor cells (MDSCs). Unbiased gene expression analysis after TLR4-Ab treatment demonstrated upregulation of genes associated with CD11b+Ly6G+ myeloid cells and downregulation of T-cell genes. Further RNA sequencing of purified, TLR4-Ab-treated CD11b+ cells showed significant upregulation of genes associated with bone marrow-derived CD11b+ cells and innate immune system genes. TLR4-Ab significantly increased percentages and numbers of CD11b+ cells. TLR4-Ab-induced CD11b+ cells, derived ex vivo from TLR4-Ab-treated mice, suppress T cells, and TLR4-Ab-conditioned bone marrow cells suppress acute T1D when transferred into acutely diabetic mice. Thus, the TLR4-Ab-induced CD11b+ cells, by the currently accepted definition, are MDSCs able to reverse T1D. To understand the TLR4-Ab mechanism, we compared TLR4-Ab with TLR4 agonist lipopolysaccharide (LPS), which cannot reverse T1D. TLR4-Ab remains sequestered at least 48 times longer than LPS within early endosomes, alters TLR4 signaling, and downregulates inflammatory genes and proteins, including nuclear factor-κB. TLR4-Ab in the endosome, therefore, induces a sustained, attenuated inflammatory response, providing an ideal "second signal" for the activation/maturation of MDSCs that can reverse acute T1D.

Neutrophilokines and the morphofunctional state of circulating neutrophils in ovarian tumors

There is currently no clear understanding of the molecular participants providing cytotoxic and/or cytostatic activity of neutrophils (Nph) in relation to tumor cells. Cytokines produced by neutrophils are necessary for their paracrine and autocrine interactions with surrounding cells. In order to assess the effect of regulatory neutrophilokines on the morphofunctional state of circulating Nph in benign ovarian tumors and ovarian cancer, the ELISA method was used to assess the level of IL-4, IL-6, IL-10, IL-8, IL-18, IFNγ, MCP-1 and MMP-1in neutrophils, expression of CD11b, CD63, CD16, CD95. Determined the rigidity of the membrane and the ability of neutrophils to form NET. Statistical processing of the obtained data was carried out using the software Statistica 13.0, Jamovi 1.6.5.0. It was found that in ovarian cancer the rigidity of neutrophils depends on the level of IL-10, MCP-1, IL-18, IL-8, IL-4, IFNγ, IL-6 in neutrophils. The method of multiple regression revealed the dependence of the ability to form NETs on the level of IL-4 and IL-6 in Nph. Revealed an inverse correlation between the rigidity of the membrane Nph and their ability to form traps in ovarian cancer. In benign ovarian tumors, a noticeable direct correlation was found between the rigidity of the neutrophil membrane and the adhesive marker CD11b. In ovarian cancer, a correlation was found between the rigidity of the Nph membrane and the CD63 degranulation marker. At benign ovarian tumors, no correlations were found between the number of activated neutrophils and the level of intracellular cytokines in Nph. In ovarian cancer, correlations were found between the number of CD11b+Nph and the level of IL-6, IL-8 in them; between the amount of CD63, CD95 and intracellular IL-8. The amount of CD16+Nph correlated with the level of MMP-1 and IL-8 in Nph. The amount of CD95+Nph correlated with the level of IL-18 in Nph. Thus, the change in the level of neutrophilokines in benign ovarian tumors did not correlate with changes in the ability to NETosis, expression of activation markers, but was accompanied by an increase in the rigidity of the membrane of circulating neutrophils. In ovarian cancer, an increase in IL-8 correlated with a decrease in CD16 expression and an increase in CD63; a decrease in CD16 correlated with an increase in MMP-1. An increase in membrane rigidity in ovarian cancer was associated with changes in all considered neutrophilokines (IL-4, IL-6, IL-8, IL-10, IL-18, MCP-1, IFNγ). The combination of IL-4, IL-6, IL-18 indices, NET number and membrane rigidity of circulating Nph (according to the results of multivariate analysis) can be used for differential diagnosis of ovarian cancer.

Abstract 1756: The roles of Regnase-1 in pancreatic cancer development and progression

Abstract Background: Regnase-1 is a RNA-degrading enzyme that plays an important role in the regulation of inflammation by degrading mRNA of inflammation-related genes. Although inflammation is known to promote pancreatic carcinogenesis, involvement of Regnase-1 in this process has never been studied. In this study, we thus investigated the role of Regnase-1 in pancreatic cancer development and progression. Methods: We examined the expression levels of Regnase-1 in the pancreas of chronic pancreatitis by repeated administration of caerullein in mice. We sought for a regulator of the Regnase-1 expression in Panc-1 pancreatic cancer cell line. We assessed the biological phenotypes caused by siRNA-mediated Regnase-1 inhibition in multiple pancreatic cancer cell lines, including cell proliferation, migration, apoptosis and cytokine expressions. We generated pancreas-specific Kras-activated and Regnase-1-deficient (PDX1-Cre; Kras G12D KI Regnase-1 fl/fl) mice (PKR mice) and examined these phenotypes. We assessed the relationship between the expression levels of Regnase-1 in pancreatic tumor tissue and prognosis in 39 pancreatic cancer patients who underwent curative surgical resection. Results: The expression levels of Regnase-1 significantly decreased in the murine pancreas of chronic pancreatitis together with the increase in IL-1β. The expression levels of Regnase-1 was downregulated by IL-1β administration in Panc-1 cells. siRNA-mediated Rengase-1 Inhibition significantly promoted cell proliferation, migration and inhibited apoptosis with Bcl-xL upregulation in Panc-1 cells. Rengase-1 Inhibition also upregulated a variety of chemokines involved in the recruitment and activation of myeloid-derived suppressor cells (MDSCs) including CXCL1, CXCL2, CXCL8, CSF2 and CSF3 in Panc1cells. All PKR mice developed pancreatic tumors and became moribund by 3 months of age. We observed massive infiltration of CD11b+Ly6g+Ly6c- PMN-MDSCs and significant upregulation of Arg1, Nos2, Nox2, and S100a8/9 mRNA levels, which are suppressors of anti-tumor immunity known to be secreted from MDSCs. Surgically-resected pancreatic cancer patients with low levels of intra-tumor Regnase-1 expression had significantly shorter recurrence-free and overall survival than those with high levels of Regnase-1. Intratumor Regnase-1 expression levels were negatively correlated with the number of CD11b+ cell infiltration. Conclusion: Inflammation-mediated downregulation of Regnase-1 may contribute to pancreatic cancer development and progression in cell-autonomous and -heteronomous manners. Citation Format: Junya Okabe, Takahiro Kodama, Tetsuo Takehara. The roles of Regnase-1 in pancreatic cancer development and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1756.

Neural precursor cells are decreased in the hippocampus of the delayed carbon monoxide encephalopathy rat model

The pathophysiology of delayed carbon monoxide (CO) encephalopathy remains unclear. In this study, the effects of CO exposure on the dentate gyrus (DG) were investigated in a Wistar rat model by histochemical and molecular methods. Model rats showed significant cognitive impairment in the passive-avoidance test beginning 7 days after CO exposure. Immunohistochemistry showed that compared to the control, the cell number of SRY (sex-determining region Y)-box 2 (SOX2)+/brain lipid binding protein (BLBP)+/glial fibrillary acidic protein (GFAP)+ cells in the DG was significantly less, but the number of SOX2+/GFAP− cells was not, reflecting a decreased number of type 1 and type 2a neural precursor cells. Compared to the control, the numbers of CD11b+ cells and neuron glial antigen 2+ cells were significantly less, but the number of SOX2−/GFAP+ cells was not. Flow cytometry showed that the percent of live microglial cells isolated from the hippocampus in this CO rat model was significantly lower than in controls. Furthermore, mRNA expression of fibroblast growth factor 2 and glial cell-derived neurotrophic factor, which are neurogenic factors, was significantly decreased in that area. We conclude that, in this rat model, there is an association between delayed cognitive impairment with dysregulated adult hippocampal neurogenesis and glial changes in delayed CO encephalopathy.