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The expression of CD14 in monocytic cells is elevated in atherosclerotic lesions where 7-oxyterols are abundant. However, it remains unknown whether atheroma-relevant 7-oxysterols are involved in receptor expression. Therefore, we investigated the effects of 7α-hydroxycholesterol (7αOHChol), 7β-hydroxycholesterol (7βOHChol), and 7-ketocholesterol (7K) on CD14 levels in THP-1 cells. The three 7-oxysterols increased CD14 transcript levels at a distinct time point, elevated cellular CD14 protein levels, and promoted the release of soluble CD (sCD14) from THP-1 cells. Our data revealed that CD14 expression was most strongly induced after treatment with 7αOHChol. Moreover, 7αOHChol alone upregulated membrane-bound CD14 levels and enhanced responses to lipopolysaccharides, as determined by CCL2 production and monocytic cell migration. The 7-oxysterols also increased the gelatinolytic activity of MMP-9, and a cell-permeable, reversible MMP-9 inhibitor, MMP-9 inhibitor I, significantly impaired sCD14 release. These results indicate that 7-oxysterols differentially induce CD14 expression in vascular cells and contribute to the monocytic cell expression of CD14 via overlapping, but distinct, mechanisms.

Leucine-rich repeat (LRR) is a structural motif has important recognition function in immune receptors, such as Tolls and NOD-like receptors (NLRs). The immune-related LRR proteins can be divided into two categories, LRR-containing proteins and LRR-only proteins. The latter contain LRR motifs while they are without other functional domains. However, the functional mechanisms of the LRR-only proteins were still unclear in invertebrates. Here, we identified a gene encoding a secretory LRR-only protein, which possessed similarity with vertebrate CD14 and was designated as LvCD14L, from the Pacific whiteleg shrimp Litopenaeus vannamei. Its transcripts in shrimp hemocytes were apparently responsive to the infection of Vibrio parahaemolyticus. Knockdown of LvCD14L with dsRNA resulted in significant increase of the viable bacteria in the hepatopancreas of shrimp upon V. parahaemolyticus infection. Further functional studies revealed that LvCD14L could bind to microorganisms’ PAMPs, showed interaction with LvToll1 and LvToll2, and regulated the expression of LvDorsal and LvALF2 in hemocytes. These results suggest that LvCD14L functions as a pattern recognition receptor and activates the NF-κB pathway through interaction with LvTolls. The present study reveals a shrimp LvCD14L-Tolls-NF-κB signaling pathway like the CD14/TLR4/NF-κB signaling pathway in mammalians, which enriches the functional mechanism of secretory LRR-only immune receptors during pathogens infection in invertebrates.

Background Recurrent respiratory syncytial virus (RSV) infection requiring hospitalization is rare and the underlying mechanism is unknown. We aimed to determine the role of CD14-mediated immunity in the pathogenesis of recurrent RSV infection. Methods We performed genotyping and longitudinal immunophenotyping of the first patient with a genetic CD14 deficiency who developed recurrent RSV infection. We analyzed gene expression profiles and interleukin (IL)-6 production by patient peripheral blood mononuclear cells in response to RSV pre- and post-fusion (F) protein. We generated CD14-deficient human nasal epithelial cells cultured at air-liquid interface (HNEC-ALI) of patient-derived cells and after CRISPR-based gene editing of control cells. We analyzed viral replication upon RSV infection. Results Sanger sequencing revealed a homozygous single-nucleotide deletion in CD14, resulting in absence of the CD14 protein in the index patient. In vitro, viral replication was similar in wild-type and CD14−/− HNEC-ALI. Loss of immune cell CD14 led to impaired cytokine and chemokine responses to RSV pre- and post-F protein, characterized by absence of IL-6 production. Conclusions We report an association of recurrent RSV bronchiolitis with a loss of CD14 function in immune cells. Lack of CD14 function led to defective immune responses to RSV pre- and post-F protein without a change in viral replication.

Background and AimsHistone deacetylase inhibitors [HDACi] exert potent anti-inflammatory effects. Because of the ubiquitous expression of HDACs, clinical utility of HDACi is limited by off-target effects. Esterase-sensitive motif [ESM] technology aims to deliver ESM-conjugated compounds to human mononuclear myeloid cells, based on their expression of carboxylesterase 1 [CES1]. This study aims to investigate utility of an ESM-tagged HDACi in inflammatory bowel disease [IBD].MethodsCES1 expression was assessed in human blood, in vitro differentiated macrophage and dendritic cells, and Crohn’s disease [CD] colon mucosa, by mass cytometry, quantitative polymerase chain reaction [PCR], and immunofluorescence staining, respectively. ESM-HDAC528 intracellular retention was evaluated by mass spectrometry. Clinical efficacy of ESM-HDAC528 was tested in dextran sulphate sodium [DSS]-induced colitis and T cell transfer colitis models using transgenic mice expressing human CES1 under the CD68 promoter.ResultsCES1 mRNA was highly expressed in human blood CD14+ monocytes, in vitro differentiated and lipopolysaccharide [LPS]-stimulated macrophages, and dendritic cells. Specific hydrolysis and intracellular retention of ESM-HDAC528 in CES1+ cells was demonstrated. ESM-HDAC528 inhibited LPS-stimulated IL-6 and TNF-α production 1000 times more potently than its control, HDAC800, in CES1high monocytes. In healthy donor peripheral blood, CES1 expression was significantly higher in CD14++CD16- monocytes compared with CD14+CD16++ monocytes. In CD-inflamed colon, a higher number of mucosal CD68+ macrophages expressed CES1 compared with non-inflamed mucosa. In vivo, ESM-HDAC528 reduced monocyte differentiation in the colon and significantly improved colitis in a T cell transfer model, while having limited potential in ameliorating DSS-induced colitis.ConclusionsWe demonstrate that monocytes and inflammatory macrophages specifically express CES1, and can be preferentially targeted by ESM-HDAC528 to achieve therapeutic benefit in IBD.

Relevance . Respiratory infections are the most common in the world. In order to prevent epidemics, there is a need to improve the strategies for organizing medical care and develop new approaches in order to increase the nonspecific resistance, mobilize innate immunity. Objective . The aim of this study was to investigate the effect of glucosaminylmuramyldipeptide (GMDP) on the level of expression of markers of differentiation and activation of functionally significant subpopulations of dendritic cells in peripheral blood mononuclear cells of healthy donors,the second aim was to assess the effectiveness of GMDP in the prevention of acute respiratory infections in an unfavorable epidemiological period of the COVID-19 pandemic. Materials and Methods . An open comparative study included 309 apparently healthy participants, aged 19-22 years. At the first stage of the study, 42 participants (22 female and 20 male) took the drug Licopid 1 mg for 10 days according to the instructions, 1 tablet 3 times a day in order to prevent acute respiratory infections. Peripheral blood sampling was performed before taking the drug (day 0) and the next day after the last dose of the drug (day 12). Evaluation of the expression of markers of differentiation and activation of dendritic cell subpopulations HLA-DR, CD11c, CD123, CD80, CD83, CCR7, CD3, CD14, CD20 was assessed by flow cytometry. At the same time, mRNA was isolated from mononuclear cells of perfusion blood and, after reverse transcription, the level of gene expression was determined by RT PCR. At the next stage, the effectiveness of the prophylactic use of the drug Licopid in 267 students of the Institute of Physical Culture was assessed in order to prevent acute respiratory infections in an unfavorable epidemiological period; the observation period was 12 months. Results and Discussion . A study of the relative quantitative composition of DCs in the peripheral blood of healthy donors by flow cytometry revealed the possibility of an increase in their total number, as well as subpopulations of MDC and PDC under the influence of GMDP. There was a statistically significant increase in the receptors for the chemokine CCR7, which is responsible for the recruitment of DCs to the secondary lymphoid organs. Analysis of the levels of expression of genes XCR1, CD11b , and CD103 showed a statistically significant effect of GMDP on an increase in their expression compared to the baseline level (before GMDP intake), with the mean value being higher in participants undergoing moderate exercise. It was found that the use of the drug Licopid 1mg for the purpose of preventing and reducing the seasonal incidence of acute respiratory infections at the stage of basic training of students of the Institute of Physical Culture contributed to a decrease in the incidence of acute respiratory infections within 12 months of observation after taking the drug. The number of episodes of acute respiratory infections decreased 3.7 times, while the group with 3 or more episodes of acute respiratory infections during the year, which constituted 14.5 % of participants, completely disappeared. The maximum efficiency of GMDP was observed in the track and field command, in which the number of participants who had no episodes of acute respiratory infections during the year increased by 7 times. Conclusion . Our data complement the modern understanding of the molecular mechanism of action of GMDP and substantiate the possibility of its experimental and clinical use in order to develop new strategies for organizing medical care in order to increase the nonspecific resistance of the organism.

β-glucan derived from the black yeast Aureobasidium pullulans (A. pullulans) is one of the natural products attracting attention due to its high potential for application as a functional food and pharmaceutical. Our team of researchers obtained a highly soluble, low-molecular-weight β-glucan from the fermentation culture medium of A. pullulans via mechanochemical ball milling method, that is, the low-molecular-weight A. pullulans-fermented β-D-glucan (LMW-AP-FBG). We investigated the anti-inflammatory effect of LMW-AP-FBG using lipopolysaccharide (LPS)-stimulated murine macrophages (RAW264.7 cells) in the current study. LMW-AP-FBG altered LPS-stimulated inflammatory responses by reducing the release of inflammatory mediators such as nitric oxide (NO), interleukin (IL)-1β, IL-6 and tumor necrosis factor-α. As well, the mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) signaling pathways were downregulated by LMW-AP-FBG. Furthermore, LMW-AP-FBG markedly reduced LPS-induced expression of cell surface molecules, CD14, CD86, and MHC class II, which mediate macrophage activation. These findings suggest that LMW-AP-FBG can be used as an effective immune modulator to attenuate the progression of inflammatory disease.

Aim: To demonstrate whether sCD14 and CD14 (rs2569190 A/G and rs2569191 C/T) genetic variants are associated with systemic lupus erythematosus (SLE) risk, for the first time, in Egyptian pediatrics and adolescents. Materials & methods: sCD14 concentrations were determined in plasma of 95 SLE cases and 98 healthy controls using ELISA assay. Genotyping was performed using TaqMan technology. Results: sCD14 levels were elevated in SLE. Individuals with T, CT and TT genotypes in rs2569191 were of significant risk (odds ratio = 1.471–2.035, 95% CI = 1.138–3.471) and those with combined CT+TT and haplotype GT were of higher risk of SLE (odds ratio = 1.660–1.758, 95% CI = 1.003–3.106, p

Background and aims Glucagon Like Peptide-1 Receptor (GLP-1R) activation reduces pro-inflammatory responses of human monocytes, their accumulation in the vascular wall and foam cell formation inhibiting atherosclerogenesis. This suggests that reduction of circulating GLP-1-1R positive monocytes may have pro-atherogenic effects. It is unknown whether different CD14/CD16 monocytes subsets display GLP-1R and whether their relative proportions correlate with atherosclerosis severity. We evaluated the association between GLP-1R positivity in different CD14/CD16 monocyte subsets and coronary atherosclerosis severity. Methods Relative amounts of classical (CD14+/CD16-), intermediate pro-inflammatory (CD14+/CD16+) and non-classical patrolling (CD14-/CD16+) subsets of total circulating monocytes and the proportions of GLP-1R positive monocytes in these subsets were determined in 13 control subjects and 10 dyslipidemic ischemic heart disease (IHD) patients with severe angiographic proven coronary atherosclerosis using flow cytometry analysis. Atherosclerosis severity was calculated by SYNTAX score. Results In univariable analysis, severe atherosclerosis was associated with decreased proportion of classical monocytes and two fold increased CD16+ pro-inflammatory and patrolling subsets as compared with controls (p = 0.01, p = 0.02 and p = 0.01, respectively). Frequency of GLP-1R positive monocytes was decreased in both CD16+ subsets (p = 0.02 and p = 0.05, respectively) and negatively correlated with atherosclerosis severity (r = −0.65, p = 0.005 and r = −0.44, p = 0.05, respectively). Conclusions Increased skewing of the classical monocyte population toward CD16+ pro-inflammatory and patrolling subsets accompanied by decreased in GLP-1R positivity are associated with coronary atherosclerosis severity in IHD patients with dyslipidemia. Although the effect of potential confounders cannot be ruled out, our data suggest that failure of GLP-1R-dependent anti-inflammatory/anti-atherogenic control results in innate immune system dysfunction and can promote atherosclerogenesis.

Background: Bacterial Endotoxin (lipopolysaccharide) is essential for bacterial virulence, as it has a biphasic effect which is either harmful and leads to aseptic shock and death or assists the body defense mechanisms as it stimulates B-cells activation. Many studies noted that LPS do their action through activation of CD14/ TLR4 pathways, which occur mainly in liver cells, including Kupffer cells, hepatocytes, and liver sinusoidal endothelial cells, which are responsible for cytokines releases and appearing the good or bad LPS effect. Aim: The current study aimed to disclose the expression changes in the profile of innate immunological receptors TLR4 and CD14 in rats' livers after stimulation with LPS. Methods: Ten groups of male Wistar albino rats were used to study the effects of two types of LPS [extracted LPS from the local strain of E. coli (ELPS) and standard E. coli (SLPS)], these were given by using different doses (5 mg/kg and 100 µg/kg), the LPS was injected either Intravenously or intraperitoneally. The TLR4 and CD14 mRNA expression pattern was estimated using qPCR after 6, 12, and 24 hours post-injection. Results: The results show that there is a negative effect of ELPS on liver CD14 and TLR4 regardless of dose and route of administration. On the other hand, the SLPS has an up regulatory impact on liver gene expression. Also, different time shows no effect on the gene expression of the two genes. Conclusion: This study concludes that both LPS types used were able to stimulate the CD14 and TLR 4 gene expression in the liver in different doses and routes of injection. Also, this study showed the possibility of using the ELPS as an immunomodulator in rats.

BACKGROUND We have previously shown that the initiation of antiretroviral therapy (ART) is associated with a decrease in skeletal muscle density (greater fat accumulation), suggesting that gains in lean body mass seen in many ART studies may reflect gains in low quality, fatty muscle. Here, we explore whether skeletal muscle density and area are associated with markers of inflammation and immune activation. METHODS ART-naive people with HIV were randomized to raltegravir or ritonavir-boosted atazanavir or darunavir, each with tenofovir disoproxil fumarate/emtricitabine. Abdominal computed tomography scans from baseline and week 96 were reanalyzed for psoas density and area and correlations explored with inflammation [interleukin-6 (IL-6) and high-sensitivity C-reactive protein] and immune activation [soluble CD14 (sCD14), soluble CD163 (sCD163), and %CD38+HLADR+ on CD4+ or CD8+ T cells]. RESULTS Two hundred twenty-two participants had available inflammation/immune activation markers and paired computed tomography scans. At baseline, lower psoas density (greater fat) correlated with higher IL-6 (r = -0.26, P < 0.001) and sCD163 (r -0.15, P = 0.03) and lower lean psoas area correlated with higher IL-6, high-sensitivity C-reactive protein, sCD14, sCD163, and %CD38+HLADR+ on CD4+ T cells (r = -0.30-0.13; all P ≤ 0.05). From baseline to week 96, greater percent decrease in total psoas density (more fat) correlated with greater increase in IL-6 (r = -0.14; P = 0.04); greater % decrease in lean psoas area correlated greater increases in IL-6, sCD14, sCD163, and %CD38+HLADR+ on CD8+ T cells (r = -0.15 to -0.18; all P < 0.04). CONCLUSIONS Greater fat infiltration within the psoas muscle (lower density) and greater loss in lean psoas muscle area were associated with higher inflammation and immune activation, which may portend important effects on muscle function and cardiometabolic risk.

Objective：Macrophages are the main source of inflammatory mediators and play important roles in the pathogenesis of rheumatoid arthritis (RA). Interleukin-21 (IL-21) regulates both innate and adaptive immune responses and exerts major effects on inflammatory responses that promote the development of RA. However, its effect on macrophage polarisation remains unclear.Methods：CD14+ monocytes of the peripheral blood of Human healthy donors (HD) and RA, and macrophages of RA synovial fluid (RA-SF MΦs) were isolated. IL-21 receptor (IL-21R) was detected by flow cytometry. Cytokine production by MΦs from different sources pre-treated with IL-21 and/or LPS was measured by real-time polymerase chain reaction (RT-PCR) and ELISA. CD14+ monocytes were differentiated into M1-like and M2-like macrophages via stimulation with GM-CSF, interferon-γ (IFN-γ), and LPS or M-CSF, IL-4, and IL-13, respectively. To determine the effect of IL-21 on macrophage polarisation, macrophage phenotypes, gene expression, and cytokine secretion were detected by flow cytometry, RT-PCR, and ELISA. TLR4 and ERK1/2 were determined by western blotting.Results：IL-21 exerted different effects on LPS-mediated inflammatory responses in various derived MΦs, and inhibited macrophages polarisation to M1-like macrophages and promote their polarisation to M2-like macrophages in HD and RA. Moreover, IL-21 inhibited LPS-mediated secretion of inflammatory cytokines, probably by downregulating the ERK1/2, in RA-SF MΦs.Conclusion：For the first time, we indicated that IL-21 inhibits LPS-mediated cytokine production in RA-SF MΦs, and impairs pro-inflammatory activity of M1-like macrophages, hereby exerting anti-inflammatory effects on RA. Thus, IL-21 might not be an appropriate therapeutic target for RA.

Genetic analysis of human inborn errors of immunity has defined the contribution of specific cell populations and molecular pathways in the host defense against infection. The STAT family of transcription factors orchestrate hematopoietic cell differentiation. Patients with de novo activating mutations of STAT3 present with multiorgan autoimmunity, lymphoproliferation, and recurrent infections. We conducted a detailed characterization of the blood monocyte and dendritic cell (DC) subsets in patients with gain-of-function (GOF) mutations across the gene. We found a selective deficiency in circulating nonclassical CD16+ and intermediate CD16+CD14+ monocytes and a significant increase in the percentage of classical CD14+ monocytes. This suggests a role for STAT3 in the transition of classical CD14+ monocytes into the CD16+ nonclassical subset. Developmentally, ex vivo–isolated STAT3GOF CD14+ monocytes fail to differentiate into CD1a+ monocyte-derived DCs. Moreover, patients with STAT3GOF mutations display reduced circulating CD34+ hematopoietic progenitors and frequency of myeloid DCs. Specifically, we observed a reduction in the CD141+ DC population, with no difference in the frequencies of CD1c+ and plasmacytoid DCs. CD34+ hematopoietic progenitor cells from patients were found to differentiate into CD1c+ DCs, but failed to differentiate into CD141+ DCs indicating an intrinsic role for STAT3 in this process. STAT3GOF-differentiated DCs produced lower amounts of CCL22 than healthy DCs, which could further explain some of the patient pathological phenotypes. Thus, our findings provide evidence that, in humans, STAT3 serves to regulate development and differentiation of nonclassical CD16+ monocytes and a subset of myeloid DCs.

Acne is a multifactorial inflammatory dermatose that affects all age categories from teenagers to adults, resulting in important psychological impacts. Multiple hypotheses currently attempt to decrypt the physiopathology of this disease, and four main actors were identified as highly implicated in it: hyperkeratinization of the pilosebaceous follicle, hyperseborrheae, host factors (innate immunity) and skin microbiota. In this letter, we present results illustrating the impact of skin microbiota on inflammatory skin response, and how far the proper balance between each bacterial community, especially C. acnes and S. epidermidis, is crucial to maintain an appropriate inflammatory response on the skin. The data presented in this study demonstrate that within the skin microbiota, an imbalance between Cutibacterium acnes and Staphylococcus epidermidis, is able to induce the activation of inflammation-related markers such as IL-1ra, IL-6, IL-8, G-CSF and the molecules C5/C5a, soluble CD14 MIP-3beta, Serpin E1, VCAM-1 and beta-defensin-2. Moreover, S. epidermidis appears to have a more important role than C. acnes on the induction of inflammation-related markers, particularly on IL-6. This work is the basis of future in vitro studies to further understand acne physiopathology, inspiring the development of future innovative therapies based on skin microbiota modulation.

Key Points Generation, identification, isolation, and expansion of monocyte and osteoclast progenitors from human embryonic stem cells.CD11b−CD14−CD115+ cells from embryonic stem-derived cultures and peripheral blood mononuclear cells contain monocyte/osteoclast progenitors., Visual Abstract, Osteoclasts are multinuclear cells of monocytic lineage, with the ability to resorb bone. Studies in mouse have identified bone marrow clonal progenitors able to generate mature osteoclast cells (OCs) in vitro and in vivo. These osteoclast progenitors (OCPs) can also generate macrophages and dendritic cells. Interestingly, cells with equivalent potential can be detected in periphery. In humans, cells with OCP activity have been identified in bone marrow and periphery; however, their characterization has not been as extensive. We have developed reproducible methods to derive, from human pluripotent stem cells, a population containing monocyte progenitors able to generate functional OCs. Within this population, we have identified cells with monocyte and osteoclast progenitor activity based on CD11b and CD14 expression. A population double positive for CD11b and CD14 contains cells with expected osteoclastic potential. However, the double negative (DN) population, containing most of the hematopoietic progenitor activity, also presents a very high osteoclastic potential. These progenitor cells can also be differentiated to macrophage and dendritic cells. Further dissection within the DN population identified cells bearing the phenotype CD15−CD115+ as the population with highest monocytic progenitor and osteoclastic potential. When similar methodology was used to identify OCPs from human peripheral blood, we confirmed a published OCP population with the phenotype CD11b+CD14+. In addition, we identified a second population (CD14−CD11bloCD115+) with high monocytic progenitor activity that was also able to form osteoclast like cells, similar to the 2 populations identified from pluripotent stem cells.

In cultures of peritoneal macrophages (MP) of male BALB/c mice infected with Mycobacterium tuberculosis from the BCG vaccine, the expression of CD1, CD14, CD25, CD30, CD35, and CD95 receptors was studied in vitro 3 months after infection. In MP cultures from intact and infected mice, mononuclear MP predominated (96 and 92%, respectively). Bi- and trinuclear MP in MP cultures from control and infected mice constituted 4 and 8.3% of all MP, respectively. In the cultures of both groups, no obvious correlations between the number of MP expressing CD-receptors and number of nuclei in these cells were found, but the expression of CD14 receptor was more often noted. In cultures from infected animals, hypertrophied MP and enhanced (by several times) expression of all CD-receptors were observed. The increase in the expression of CD-receptor can be determined by activation of plastic processes in hypertrophied MP (in epithelioid and in numerically insignificant polynuclear MP), which is due to the phenomenon of prolonged M. tuberculosis persistence in the vacuolar apparatus of these cells.

Introduction Symptomatic juvenile idiopathic arthritis (sJIA) is an autoinflammatory disease, and monocytes/macrophages play an important role. However, which macrophage subtype plays a major role in different stages of sJIA is still unclear. This study aimed to explore macrophage subtypes in different stages of sJIA. Methods Twenty-two children with sJIA who were followed up at Shanghai Children’s Hospital from January 2018 to December 2020 were enrolled in this study. sJIA children were divided into an activity group (n = 12) and an inactivity group (n = 10). In the activity group, subjects with newly diagnosed sJIA and untreated were included; in the inactivity group, subjects with inactive sJIA meeting the 2011 ACR criteria for sJIA were recruited. Ten children with orthostatic proteinuria served as controls. Peripheral blood was collected. Flow cytometry was performed to detect macrophage subtypes: M1 (CD14+CD86+CD80+), M2a (CD14+CD206+CD301+), M2b (CD14+CD206+CD86+) and M2c (CD14+CD206+CD163+), and the contents of cytokines were also examined, including interleukins (IL) (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10 and IL-17), interferon-α, interferon-γ, and tumor necrosis-α. Results M1 marker CD80 and M2 marker CD163, CD301 were highly expressed in children with active sJIA. The majority of macrophages were M1 and M2a in the activity group (P

The timely diagnosis and treatment of post-infectious glomerulonephritis (PIGN) is currently limited by the erased and low-symptom nature of the disease, which leads to the search for informative biological markers of the disease, which can be used as immunological indicators of blood and urine. The study was carried out in order to establish the characteristic changes in the immunological parameters of blood and urine in patients with PIGN. The study included 60 patients with PIGN from among the patients, hospitalized in the nephrology department of the Republican Clinical Hospital of Health Care Ministry of the Chuvash Republic in 2015-2018. In addition to the generally accepted research methods, the patients underwent: 1) the determination of indicators of innate and acquired immune response in the blood (CD3+ -, CD3+ CD4+-, CD3+CD8+-, CD4+CD25+-, CD95+-, CD20+-, CD14+CD282+-, CD14+CD284+- cells; levels of IgG, IgA, IgM, circulating immune complexes, C3, C4) and urine (levels of IgG, IgA, IgM, C3, C4); 2) the determination of the levels of cytokines - IL-1β, Ra-IL-1β, IL-2, IL-4, IL-8, IL-10, IL-17A in blood serum and urine. The data obtained were compared with those of the group of healthy individuals. The changes in blood immunological parameters, identified in the group of patients with PIGN, indicate the activation of innate immunity (the increase in the number of CD14+TLR2+- cells) and the humoral component of adaptive immunity (the increase in the number of B-lymphocytes, hyperimmunoglobulinemia - the increase in IgM and IgA levels) against the background of the decrease in the number of T (CD3+) - lymphocytes and regulatory (CD4+CD25high) - cells, hypocomplementemia (decreased levels of C3, C4). The increase in the content of C3, IgG and IgA was found in the urine. The cytokine profile of blood in patients with PIGN was characterized by the increase in the levels of pro- and anti-inflammatory cytokines IL-1β, Ra-IL-1β, IL-2, IL-8, IL-10, IL-17A, with the exception of IL-4, which remained on the levels of healthy individuals. The cytokine profile of urine in patients was characterized by the increase in the levels of pro-inflammatory cytokines IL-1β, IL-2, IL-8, IL-17A and anti-inflammatory cytokine - IL-10, with no changes in the content of Ra-IL-1β and IL-4. The revealed features of the immunological profile of blood and urine in patients with PIGN reflect the immunopathogenetic mechanisms of this disease.

Tumor-associated macrophages (TAMs) are known to participate in osteosarcoma (OS) progression. As demonstrated in our previous research, miR-363 played a tumor inhibitory effect in OS cells via lowering the PDZ domain containing 2 (PDZD2) expression. The regulatory roles of TAMs on miR-363/PDZD2 and the internal mechanism relating to long noncoding RNA p53 upregulated regulator of P53 levels (lncRNA PURPL) are examined in this study. TAM-like macrophages were formed by inducing CD14+ peripheral blood mononuclear cells (PBMCs). The TAMs migration was detected after MG-63 cells transfected with miR-363 mimics or inhibitors. We then analyzed the regulatory activity of PURPL on miR-363 expression. We also tested the influences of PURPL overexpression/knockdown on MG-63 cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT), as well as TAMs migration. Silence in PDZD2 expression was used to confirm the effects of PURPL on MG-63 cells. We successfully induced TAM-like macrophages. MG-63 cells transfecting miR-363 mimics suppressed TAMs migration while transfecting a converse effect was seen in miR-363 inhibitor. TAMs raised PURPL expression in MG-63 cells, which was an upstream regulator of miR-363. Along with TAMs migration, PURPL overexpression promoted MG-63 cell proliferation, migration, invasion, and EMT. An opposite influence was seen due to the PURPL knockdown. The silence of PDZD2 weakened the influences of PURPL overexpression on MG-63 cells and TAMs migration. On modulating the PURPL/miR-363/PDZD2 axis, TAMs-promoted OS development might be achieved.

Background: Globally, highest hematologic malignancy in prevalence is considered chronic lymphocytic leukemia (CLL). The expression of angiopoietin-2 (Ang2) and tyrosine kinase with immunoglobulin and endothelial growth factor homology domains (Tie1), two critical components of the Ang-Tie2 pro-angiogenic pathway, in CLL cells has been demonstrated. Objective: Clarification of Tie2-expressing monocytes’ (TEMs) involvement in the pathophysiology of CLL is the goal of this study. Patients and Methods: The study was case control, which was performed on 21 CLL patients; their age ranged from 46 to 71 with a mean of 58.2 ± 6.9, in addition to 21 age and sex matched healthy control subjects. Full medical histories, clinical examinations, and laboratory tests were conducted on all individuals. The percentage of TEMs in peripheral blood was determined by flow cytometry and their phenotypic characteristics defined as CD14+/CD16+/Tie-2+ cells. Results: Compared to the control group, there was a statistically significant increase in TEMs in the cases group. Among the cases group there was a statistical significant increase in LDH, uric acid, WBCs, reticulocyte, CD 38 β2 microglobulin and TEMS and statistical significant decrease in Hb with increase Binet stage. There was a statistical significant +ve correlation between TEMs and LDH, WBCs, reticulocytes, CD38 and β2 microglobulin. Also, there was a statistical significant negative correlation between TEMs and Hb and platelets count among the cases group. TEMs percentage had sensitivity, specificity and accuracy (100%) in diagnosis of leukemia at cut off > 20.95%. Conclusion: TEMs could be a part of CLL pathogenesis which can be a predictor of disease progression alongside with other prognostic indicators.

Objective To investigate the correlation between Coronin-1 and macrophage polarization and its possible mechanism. Methods Firstly, M1 and M2 RAW246.7 macrophage cells were established, and the expression of Coronin-1 at mRNA and protein level was detected by real-time PCR and Western blotting. Then, over-expressed (Coronin-1 Plus) and knockout (Coronin-1 KO) cell lines were constructed in RAW246.7 cells, and the expression levels of inducible nitric oxide synthase (iNOS) in over-expressed, knockout and normal-expressed Coronin-1 cells were detected by real-time PCR and Western blotting. The interaction and enrichment of Coronin-1 and CD14 related proteins were analyzed by protein-protein interaction (PPI) network and Gene Ontology (GO) enrichment analysis. Real-time PCR and Western blotting were used to detect the expression level of CD14 in over-expressed, knockout and normal Coronin-1 cells, and the expression level of iNOS after treatment with 100 ng/mL LPS. Then, CD14 knockdown cell lines (Control-CD14 KD) and CD14 knockdown Coronin-1 Plus cell lines (Coronin-1 Plus-CD14 KD) were constructed. After treatment with LPS (100 ng/mL), the expression of iNOS was detected by real-time PCR and Western blotting. Results M1 and M2 macrophage models were successfully constructed, and the expression of Coronin-1 in M1 type macrophages was significantly higher than that in the Control group (P < 0.05). The mRNA expression of iNOS was significantly lower in Coronin-1 Plus group than control group (P < 0.01), and the expression of CD14 was obviously higher in Coronin-1 Plus group than Control group (P < 0.05). After LPS treatment, the expression of iNOS in Coronin-1 Plus group was significantly higher than that in Control group (P < 0.01). There was no significant difference in iNOS level of the LPS treated Control-CD14 KD and Coronin-1 Plus-CD14 KD groups with the Control group (P>0.05). Conclusion The M1 polarization of macrophages leads to higher expression of Coronin-1, but the over-expression of Coronin-1 does not directly lead to the M1 polarization of macrophages. Its over-expression can lead to up-regulation of CD14 expression, and Coronin-1 can promote LPS-induced macrophages to M1 type polarization through the CD14 pathway.

Introduction. Influence of incidental nanoparticles of industrial aerosols on occupational lung diseases development is not studied enough. As nanoparticles has properties to induce inflammation and fibrosis, it is hypothesized that they affect occupational chronic obstructive pulmonary disease (COPD) phenotype. The aim was to establish monocyte subsets, airway inflammation, clinical and functional features in occupational COPD due to aerosols containing nanoparticles exposure. Materials and methods. Study design was a single center prospective cohort observational. Subjects with occupational COPD (GOLD 2011-2021 criteria) exposed to aerosols containing nanoparticles (n=50) enrolled. Comparison group - COPD in tobacco smokers (n=50), control group - healthy people (n=50). Groups were matched by demographics and COPD duration. Nanoparticles at workplaces air were measured by inductively coupled plasma atomic emission spectrometry and by scanning electron microscopy. Of participants 26 were exposed to maximal concentrations of metal nanoparticles and 24 - of silica nanoparticles. Spirography, body pletysmography, lung diffusing capacity (DLco/Va), Doppler-ehocardiography, induced sputum cytology, COPD exacerbations assessment were done. Monocyte subsets were determined by flow cytometry. Linear regression model was used to explore relationships. Results. COPD due to aerosols containing metal nanoparticles was characterized by most severe airflow limitation, lung hyperinflation, pulmonary hypertension, most prominent decrease in DLco/Va, frequent and severe COPD exacerbations, eosinophilic inflammation. The largest proportion of «classical» CD14+CD16- monocytes subset, 96,4% (90,3%; 97,2%), high level of CCR5 expression were seen in this group. The features of COPD due to aerosols containing silica nanoparticles were substantial decrease in DLco/Va, the least airflow limitation, mild lung hyperinflation, rare COPD exacerbations, paucigranulocytic inflammation. The largest proportion of «non-classical» CD14DimCD16+ monocytes with high level of CCR2 expression revealed. Mass concentration of metal nanoparticles was associated with «classical» monocytes, (B=1,5), silica nanoparticles - with «non-classical» monocytes (B=1,4). On their turn, «classical» monocytes were associated with DLco (B=-1,6), functional residual volume (B=1,2), mean pulmonary artery pressure (B=-1,4), eosinophilic inflammation. «Non-classical» monocytes were associated with DLco (B=-1,5) and paucigranulocytic inflammation (B=1,2), p

OBJECTIVE In rheumatoid arthritis (RA), elevated serum interleukin-34 (IL-34) levels are linked with increased disease severity. IL-34 binds to 2 receptors, macrophage colony-stimulating factor receptor (M-CSFR) and syndecan 1, which are coexpressed in RA macrophages. Expression of both IL-34 and syndecan 1 is strikingly elevated in the RA synovium, yet their mechanisms of action remain undefined. This study was undertaken to investigate the mechanism of action of IL-34 in RA. METHODS To characterize the significance of IL-34 in immunometabolism, its mechanism of action was elucidated in joint macrophages, fibroblasts, and T effector cells using RA and preclinical models. RESULTS Intriguingly, syndecan 1 activated IL-34-induced M-CSFR phosphorylation and reprogrammed RA naive cells into distinctive CD14+CD86+GLUT1+ M34 macrophages that expressed elevated levels of IL-1β, CXCL8, and CCL2. In murine M34 macrophages, the inflammatory phenotype was accompanied by potentiated glycolytic activity, exhibited by transcriptional up-regulation of GLUT1, c-Myc, and hypoxia-inducible factor 1α (HIF-1α) and amplified pyruvate and l-lactate secretion. Local expression of IL-34 provoked arthritis by expanding the glycolytic F4/80-positive, inducible nitric oxide synthase (iNOS)-positive macrophage population, which in turn attracted fibroblasts and polarized Th1/Th17 cells. The cross-talk between murine M34 macrophages and Th1/Th17 cells broadened the inflammatory and metabolic phenotypes, resulting in the expansion of IL-34 pathogenicity. Consequently, IL-34-instigated joint inflammation was alleviated in RAG-/- mice compared to wild-type mice. Syndecan 1 deficiency attenuated IL-34-induced arthritis by interfering with joint glycolytic M34 macrophage and osteoclast remodeling. Similarly, inhibition of glycolysis by 2-deoxy-d-glucose reversed the joint swelling and metabolic rewiring triggered by IL-34 via HIF-1α and c-Myc induction. CONCLUSION IL-34 is a novel endogenous factor that remodels hypermetabolic M34 macrophages and facilitates their cross-regulation with T effector cells to advance inflammatory bone destruction in RA.

Introduction The low frequency of ectopic germinal center in labial salivary glands of patients with human T‐cell leukemia virus type 1 (HTLV‐1) antibody‐positive Sjögren's syndrome (SS) suggests that HTLV‐1 has some effects on follicular dendritic cells (FDCs). Methods We used flow cytometry, immunofluorescence, and enzyme‐linked immunosorbent assays (ELISAs) to investigate the direct effect of HTLV‐1 on B‐cell activating factors produced by established FDC like cells obtained from excised human tonsils. We then measured the serum B‐cell activating factor (BAFF) and C‐X‐C motif ligand (CXCL) 13 concentrations of the HTLV‐1‐seropositive SS patients and the HTLV‐1‐seronegative SS patients by ELISA. Results Among the 31 isolated specimens, 22 showed morphological characteristics of FDCs. Day 2‐cultured specimens showed expressions of CD14, CD23, CD40, intracellular adhesion molecule‐1 (ICAM‐1), and vascular cell adhesion molecule‐1. After 2 weeks, 12 of these specimens expressed ICAM‐1, FDC, and fibroblast cell marker. Intracellular BAFF and CXCL13 were constitutively expressed regardless of stimulation. After direct coculture with the HTLV‐1‐infected T‐cell line HCT‐5 or MT‐2, the BAFF and CXCL13 expressions on the FDC‐like cells were decreased in accord with the increased number of HCT‐5 and MT‐2 cells with styliform change and without HTLV‐1 Gag protein expression. Interferons upregulated the concentration of BAFF (but not CXCL13) in the culture supernatant, which showed a declining trend under the presence of HCT‐5 or MT‐2. The serum concentrations of BAFF and CXCL13 in the HTLV‐1‐seropositive SS patients were lower than those of the HTLV‐1 seronegative SS patients. Conclusions HTLV‐1 partially inhibited the BAFF and CXCL13 expressions of established FDC‐like cells., Graphical abstract Expression of B‐cell activating factor (BAFF) was upregulated by addition of interferons. Although BAFF expression was inhibited by addition of human T‐cell leukemia virus type 1 infected cell line, HCT‐5 and MT‐2, the level was not inhibited by addition of control cell line, MOLT‐4.

Dermatomyositis (DM) is a rare, systemic autoimmune disease that most frequently affects the skin, muscles, and lungs. The inflammatory infiltrate in the skin has not been fully characterized, and, in this study, we took a single-cell, unbiased approach using imaging mass cytometry. Substantial monocyte‒macrophage diversity was observed, with the CD14+ population correlating positively with Cutaneous Dermatomyositis Disease Area and Severity Index scores (P = 0.031). The T-cell compartment revealed CD4+ T, CD8+ T, and FOXP3+ T cells. Activated (CD69+) circulating memory T cells correlated positively with Cutaneous Dermatomyositis Disease Area and Severity Index scores (P = 0.0268). IFN-β protein was highly upregulated in the T-cell, macrophage, dendritic cell, and endothelial cell populations of DM skin. Myeloid dendritic cells expressed phosphorylated peroxisome proliferator‒activated receptor γ, phosphorylated IRF3, IL-4, and IL-31, and their quantity correlated with itch as measured in Skindex-29. Plasmacytoid dendritic cells colocalized with IFN-γ in addition to the known colocalization with IFN-β. Nuclear phosphorylated peroxisome proliferator‒activated receptor γ expression was found in the DM endothelium. Imaging mass cytometry allows us to characterize single cells in the immune cell population and identify upregulated cytokines and inflammatory pathways in DM. These findings have important implications for the development of future targeted therapies for DM.

Introduction The development of pancreatic ductal adenocarcinoma (PDAC) is closely tied with the immune system. C‐C motif chemokine ligands (CCL) were proved to lead to immune recruitment and training. Thus, we reckoned CCL2 to be the kernel of immune suppression in PDAC tissues. Methods We compared normal pancreatic tissues with PDAC tissues according to The Cancer Genome Atlas (TCGA) and clinical samples. Flow cytometry was used to identify M‐MDSCs. We further demonstrated immune suppression of M‐MDSCs according to proliferation rates of CD8+ T cells/CD4+ T cells. Levels of reactive oxygen species (ROS) and Arginase were also tested by flow cytometry, enzyme‐linked immunosorbent assay, and western blot analysis. We also analyzed the specific mechanisms by cluster analysis after CCL2 stimulating M‐MDSCs. Results We found that CCL2 highly increased in PDAC tissues. CCL2 is positively related to CD33 and CD14, markers of monocytic myeloid‐derived suppressor cells (M‐MDSCs). We have demonstrated that CCL2 recruited M‐MDSCs into PDAC tissues both in vitro and in vivo. M‐MDSCs recruitment is accompanied by sustained immune suppression. Furthermore, we have found that M‐MDSCs impeded T cell proliferation and produced high levels of ROS and Arginase, which can be enhanced by CCL2. Mechanistically, CCL2 stimulated M‐MDSCs led to a significant upregulation of genes, a large part of which accumulated in the mitogen‐activated protein kinase signaling pathway. Treatment of aloesin, MAPK signaling inhibitor, relieved the associated immunosuppressive phenotype induced by CCL2. Conclusions Our study indicates that PDAC cells produced CCL2, which promoted localized M‐MDSC recruitment and immune suppression, thereby promoting tumor progression., Our data indicate that CCL2 performs a significant role in PDAC progression by recruiting and activating M‐MDSCs. Targeting MDSCs is able to improve antitumoral immunological responses providing with potential applicability of immune‐based combination therapies against an extensive spectrum of solid tumors. These miscellaneous approaches might prove available for tumor therapeutics against solid carcinomas in which M‐MDSCs perform a major role in immune evasion of tumors. These results are promising and need further assessment of the M‐MDSCs‐targeting combination or vaccination approaches for the whole therapeutic capacity of these tactics in PDAC and other carcinomas, Highlights CCL2 is significantly upregulated in colon adenocarcinoma CCL2 influences M‐MDSC recruitment and functionality in colon adenocarcinoma CCL2 stimulates immune‐suppressive functions of M‐MDSC by activating MAPK signaling

BACKGROUND: Identifying the immune cells involved in coronavirus disease 2019 (COVID-19) disease progression and the predictors of poor outcomes is important to manage patients adequately. METHODS: This prospective observational cohort study enrolled 48 patients with COVID-19 hospitalized in a tertiary hospital in Oman and 53 non-hospitalized patients with confirmed mild COVID-19. RESULTS: Hospitalized patients were older (58 years vs 36 years, P < 0.001) and had more comorbid conditions such as diabetes (65% vs 21% P < 0.001). Hospitalized patients had significantly higher inflammatory markers (P < 0.001): C-reactive protein (114 vs 4 mg/l), interleukin 6 (IL-6) (33 vs 3.71 pg/ml), lactate dehydrogenase (417 vs 214 U/l), ferritin (760 vs 196 ng/ml), fibrinogen (6 vs 3 g/l), D-dimer (1.0 vs 0.3 µg/ml), disseminated intravascular coagulopathy score (2 vs 0), and neutrophil/lymphocyte ratio (4 vs 1.1) (P < 0.001). On multivariate regression analysis, statistically significant independent early predictors of intensive care unit admission or death were higher levels of IL-6 (odds ratio 1.03, P = 0.03), frequency of large inflammatory monocytes (CD14+CD16+) (odds ratio 1.117, P = 0.010), and frequency of circulating naive CD4+ T cells (CD27+CD28+CD45RA+CCR7+) (odds ratio 0.476, P = 0.03). CONCLUSION: IL-6, the frequency of large inflammatory monocytes, and the frequency of circulating naive CD4 T cells can be used as independent immunological predictors of poor outcomes in COVID-19 patients to prioritize critical care and resources.

BACKGROUND AND AIMS Recent development of multiple treatments for human hepatocellular carcinoma (HCC) has allowed for the selection of combination therapy to enhance the effectiveness of monotherapy. Optimal selection of therapies is based on both HCC and its microenvironment. Therefore, it is critical to develop and validate preclinical animal models for testing clinical therapeutic solutions. APPROACH AND RESULTS We established cell line-based or patient-derived xenograft-based humanized-immune-system mouse models with subcutaneous and orthotopic HCC. Mice were injected with human-specific antibodies (Abs) to deplete human immune cells. We analyzed the transcription profiles of HCC cells and human immune cells by using real-time PCR and RNA sequencing. The protein level of HCC tumor cells/tissues or human immune cells was determined by using flow cytometry, western blotting, and immunohistochemistry. The HCC tumor size was measured after single, dual-combination, and triple-combination treatment using N-(1',2-Dihydroxy-1,2'-binaphthalen-4'-yl)-4-methoxybenzenesulfonamide (C188-9), bevacizumab, and pembrolizumab. In this study, human immune cells in the tumor microenvironment were strongly selected and modulated by HCC, which promoted the activation of the IL-6/Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in tumor cells and led to augmented HCC proliferation and angiogenesis by releasing angiogenic cytokines in humanized-immune-system mice with HCC. In particular, intratumor human cluster of differentiation-positive (hCD14+ ) cells could produce IL-33 through damage-associated molecular pattern/Toll-like receptor 4/activator protein 1, which up-regulated IL-6 in other intratumor immune cells and activated the JAK2/STAT3 pathway in HCC. Specific knockdown of the CD14 gene in human monocytes could impair IL-33 production induced by cell lysates. Subsequently, we evaluated the in vivo anti-HCC effect of C188-9, bevacizumab, and pembrolizumab. The results showed that the anti-HCC effect of triple-combination therapy was superior to that of single or dual treatments. CONCLUSIONS Humanized-immune-system HCC mouse models are suitable for identifying targets from cancer and immune components and for testing combinational therapies.

In dry age-related macular degeneration (AMD), inflammation plays a key role in disease pathogenesis. Innate immune cells such as microglia and neutrophils infiltrate the sub-retinal space (SRS) to induce chronic inflammation and AMD progression. But a major gap in our understanding is how these cells interact with each other in AMD. Here, we report a novel concept of how dynamic interactions between microglia and neutrophils contribute to AMD pathology. Using well-characterized genetically engineered mouse models as tools, we show that in the diseased state, retinal pigmented epithelial (RPE) cells trigger pro-inflammatory (M1) transition in microglia with diminished expression of the homeostatic marker, CX3CR1. Activated microglia localize to the SRS and regulate local neutrophil function, triggering their activation and thereby inducing early RPE changes. Ligand receptor (LR)-loop analysis and cell culture studies revealed that M1 microglia also induce the expression of neutrophil adhesion mediators (integrin β1/α4) through their interaction with CD14 on microglia. Furthermore, microglia-induced neutrophil activation and subsequent neutrophil-mediated RPE alterations were mitigated by inhibiting Akt2 in microglia. These results suggest that the Akt2 pathway in microglia drives M1 microglia-mediated neutrophil activation, thereby triggering early RPE degeneration and is a novel therapeutic target for early AMD, a stage without treatment options.

Cilj istraživanja: Cilj ovog istraživanja je utvrditi kako trening različitog intenziteta (ispod ili iznad bazalnog metabolizma) utječe na monocitne subpopulacije na uzorku sportaša rekreativaca. Materijali i metode: Uzorci krvi svih sudionika uzeti su za analizu 30 minuta prije i nakon treninga. Uzorak je tretiran reagensom za blokiranje Fc receptora te je inkubirana 20 minuta u mraku na 25°C s 4 μL anti-humanih CD14s PerCP-Cy5.5 konjugiranih antitijela i 5 μL fikoeritrinom konjugiranih antitijela reaktivnih na ljudski CD16. Nakon lize crvenih krvnih stanica otopinom za lizu, stanice su analizirane na protočnom citometru. Mjereni su također i uzorci neobojanih stanica i obrađeni su kao negativne kontrole. Podaci prikupljeni citometrom analizirani su pomoću softvera FlowLogic. Rezultati: Kao rezultat istraživanja dobili smo podatak da tjelesna aktivnost ima utjecaj na aktivaciju protuupalne te upalne monocitne skupine. Povećanjem intenziteta tjelesne aktivnosti opažen je porast broja monocitnih skupina. Također rezultati govore da vrijeme uzorkovanja krvi i broj ispitanika utječu na rezultate. Zaključak: Trening različitog intenziteta kod sportaša rekreativaca (ispod ili iznad bazalnog metabolizma) utjecao je na porast broja upalnih i protuupalnih monocita, ali je za bolje razumijevanje utjecaja intenziteta potrebno uzeti u obzir vrijeme uzorkovanja krvi i veći uzorak., The aim of this study: The aim of this research is to determine how training at different intensities (below or above basal metabolism) affects monocyte subpopulations in a sample of recreational athletes. Methods: Blood samples of all participants were taken for analysis 30 minutes before and after training. The sample was treated with Fc receptor blocking reagent and incubated for 20 minutes in the dark at 25°C with 4 μL of anti-human CD14s PerCP-Cy5.5 conjugated antibodies and 5 μL of phycoerythrin-conjugated antibodies reactive to human CD16. After the lysis of red blood cells with lysis solution, the cells were analyzed on a flow cytometer. Samples of unstained cells were also measured and processed as negative controls. Data collected by cytometer were analyzed using FlowLogic software. Results: As a result of the research, we obtained an information that physical activity has an impact on the activation of anti-inflammatory and inflammatory monocyte groups. By increasing the intensity of physical activity, an increase in the number of monocyte groups was observed. The results also tell that the time of blood sampling and the number of subjects have an influence on results. Conclusion: Training of different intensity in recreational athletes (below or above basal metabolism) affects the increase in the number of inflammatory and anti-inflammatory monocytes, but for a better understanding of intensity influence, it is necessary to take into account the time of blood sampling and a larger sample.

Infections with Gram-negative bacteria are still among the leading causes of infection-related deaths. Several studies suggest that the chalcone xanthohumol (XN) found in hop (Humulus lupulus) possesses anti-inflammatory effects. In a single-blinded, placebo controlled randomized cross-over design study we assessed if the oral intake of a single low dose of 0.125 mg of a XN derived through a XN-rich hop extract (75% XN) affects lipopolysaccharide (LPS)-induced immune responses in peripheral blood mononuclear cells (PBMCs) ex vivo in normal weight healthy women (n = 9) (clinicaltrials.gov: NCT04847193) and determined associated molecular mechanisms. LPS-stimulation of PBMCs isolated from participants 1 h after the intake of the placebo for 2 h resulted in a significant induction of pro-inflammatory cytokine release which was significantly attenuated when participants had consumed XN. The XN-dependent attenuation of proinflammatory cytokine release was less pronounced 6 h after the LPS stimulation while the release of sCD14 was significantly reduced at this timepoint. The LPS-dependent activation of hTLR4 transfected HEK293 cells was significantly and dose-dependently suppressed by the XN-rich hop extract which was attenuated when cells were co-challenged with sCD14. Taken together, our results suggest even a one-time intake of low doses of XN consumed in a XN-rich hop extract can suppress LPS-dependent stimulation of PBMCs and that this is related to the interaction of the hop compound with the CD14/TLR4 signaling cascade.

Immunomodulation by adipose-tissue-derived stem cells (ADSCs) is of special interest for the alleviation of damaging inflammatory responses in central nervous system injuries. The present study explored the effects of cerebrospinal fluid (CSF) from traumatic brain injury (TBI) patients on this immunomodulatory potential of ADSCs. CSF conditioning of ADSCs increased messenger RNA levels of both pro- and anti-inflammatory genes compared to controls. Exposure of phorbol-12-myristate-13-acetate-differentiated THP1 macrophages to the secretome of CSF-conditioned ADSCs downregulated both proinflammatory (cyclooxygenase-2, tumor necrosis factor alpha) and anti-inflammatory (suppressor of cytokine signaling 3, interleukin-1 receptor antagonist, and transforming growth factor beta) genes in these cells. Interleukin-10 expression was elevated in both naive and conditioned secretomes. ADSC secretome treatment, further, induced macrophage maturation of THP1 cells and increased the percentage of CD11b+, CD14+, CD86+, and, to a lesser extent, CD206+ cells. This, moreover, enhanced the phagocytic activity of CD14+ and CD86+ cells, though independently of pre-conditioning. Secretome exposure, finally, also induced a reduction in the percentage of CD192+ adherent cells in cultures of peripheral blood mononuclear cells (PBMCs) from both healthy subjects and TBI patients. This limited efficacy (of both naive and pre-conditioned secretomes) suggests that the effects of lymphocyte-monocyte paracrine signaling on the fate of cultured PBMCs are strongest upon adherent cell populations.

MΦs display remarkable plasticity and the ability to activate diverse responses to a host of intracellular and external stimuli. Despite extensive characterization of M1 MΦs and a broad set of M2 MΦs, comprehensive characterization of functional phenotype and associated metabotype driving this diverse MΦ activation remains. Herein, an ex vivo model was utilized to produce 6 MΦ functional phenotypes. Isolated CD14+ PBMCs were differentiated into resting M0 MΦs, and then polarized into M1 (IFN-γ/LPS), M2a (IL-4/IL-13), M2b (IC/LPS), M2c (IL-10), and M2d (IL-6/LIF) MΦs. The MΦs were profiled using a bioanalyte matrix of 4 cell surface markers, ∼50 secreted proteins, ∼800 expressed myeloid genes, and ∼450 identified metabolites relative to M0 MΦs. Signal protein and expressed gene profiles grouped the MΦs into inflammatory (M1 and M2b) and wound resolution (M2a, M2c, and M2d) phenotypes; however, each had a unique metabolic profile. While both M1 and M2b MΦs shared metabotype profiles consistent with an inflammatory signature; key differences were observed in the TCA cycle, FAO, and OXPHOS. Additionally, M2a, M2c, and M2d MΦs all profiled as tissue repair MΦs; however, metabotype differences were observed in multiple pathways including hexosamine, polyamine, and fatty acid metabolism. These metabolic and other key functional distinctions suggest phagocytic and proliferative functions for M2a MΦs, and angiogenesis and ECM assembly capabilities for M2b, M2c, and M2d MΦs. By integrating metabolomics into a systems analysis of MΦ phenotypes, we provide the most comprehensive map of MΦ diversity to date, along with the global metabolic shifts that correlate to MΦ functional plasticity in these phenotypes.

Functionally, cluster of differentiation 14 (CD14) is a co-receptor of the complex formed by lipopolysaccharide (LPS) and LPS-binding protein expressed on the membrane of a variety of cells. However, CD14 can be shed from the cell membrane into the circulation as soluble CD14 (sCD14) upon cell activation. Previously, our group reported that elevated sCD14 serum levels were associated with the clinical and laboratory findings in the context of visceral leishmaniasis (VL), but not in the context of LPS stimulation or bacterial infection. In the present study, we investigated the secretion dynamics of sCD14 in the context of Leishmania infantum (syn. L. chagasi) in vitro infection. Macrophages from treated VL patients and delayed-type hypersensitivity positive (DTH+) subjects were infected with L. infantum (syn. L. chagasi) promastigotes, and the infection index was evaluated (number of amastigotes per 100 infected macrophages). Additionally, the levels of sCD14, Inteleukin (IL)10, IL-6 and tumour necrosis factor alpha (TNF-α) were measured in the culture supernatants using the Luminex assay. Interestingly, the release of sCD14 was inversely correlated with the L. infantum (syn. L. chagasi) infection index. Of note, the release of sCD14 was upregulated and downregulated in the context of infected macrophages from DTH+ subjects and treated VL patients, respectively. Additionally, we also observed that the levels of sCD14 in the culture supernatants were positively correlated with the levels of TNF-α, IL-6 and IL-10. Therefore, our data suggest that macrophages from treated VL patients and DTH+ subjects respond differently to L. infantum (syn. L. chagasi) infection in the context of the release of sCD14; therefore, the release of sCD14 may be associated with the outcome of VL.

Background Acute radiation syndrome (ARS) is caused by acute exposure to ionizing radiation that damages multiple organ systems but especially the bone marrow (BM). We have previously shown that human macrophages educated with exosomes from human BM-derived mesenchymal stromal cells (MSCs) primed with lipopolysaccharide (LPS) prolonged survival in a xenogeneic lethal ARS model. The purpose of this study was to determine if exosomes from LPS-primed MSCs could directly educate human monocytes (LPS-EEMos) for the treatment of ARS. Methods Human monocytes were educated by exosomes from LPS-primed MSCs and compared to monocytes educated by unprimed MSCs (EEMos) and uneducated monocytes to assess survival and clinical improvement in a xenogeneic mouse model of ARS. Changes in surface molecule expression of exosomes and monocytes after education were determined by flow cytometry, while gene expression was determined by qPCR. Irradiated human CD34+ hematopoietic stem cells (HSCs) were co-cultured with LPS-EEMos, EEMos, or uneducated monocytes to assess effects on HSC survival and proliferation. Results LPS priming of MSCs led to the production of exosomes with increased expression of CD9, CD29, CD44, CD146, and MCSP. LPS-EEMos showed increases in gene expression of IL-6, IL-10, IL-15, IDO, and FGF-2 as compared to EEMos generated from unprimed MSCs. Generation of LPS-EEMos induced a lower percentage of CD14+ monocyte subsets that were CD16+, CD73+, CD86+, or CD206+ but a higher percentage of PD-L1+ cells. LPS-EEMos infused 4 h after lethal irradiation significantly prolonged survival, reducing clinical scores and weight loss as compared to controls. Complete blood counts from LPS-EEMo-treated mice showed enhanced hematopoietic recovery post-nadir. IL-6 receptor blockade completely abrogated the radioprotective survival benefit of LPS-EEMos in vivo in female NSG mice, but only loss of hematopoietic recovery was noted in male NSG mice. PD-1 blockade had no effect on survival. Furthermore, LPS-EEMos also showed benefits in vivo when administered 24 h, but not 48 h, after lethal irradiation. Co-culture of unprimed EEMos or LPS-EEMos with irradiated human CD34+ HSCs led to increased CD34+ proliferation and survival, suggesting hematopoietic recovery may be seen clinically. Conclusion LPS-EEMos are a potential counter-measure for hematopoietic ARS, with a reduced biomanufacturing time that facilitates hematopoiesis.

Objective Antipsychotics, in particular olanzapine, are first-line medications for schizophrenia. The prefrontal cortex (PFC) is an important region for antipsychotics' therapeutic effects. The PFC inflammatory and immune pathways are associated with schizophrenia pathogenesis. However, the effect of antipsychotics on the inflammatory and immune pathways in the PFC remains unclear. We aimed to examined the time-dependent effect of olanzapine on inflammatory and immune markers in the PFC of rats. Since the inflammatory and immune pathways are related to endoplasmic reticulum (ER) stress, we further investigated whether or not olanzapine-induced inflammation and immune responses were related to ER stress. Methods Expression of pro-inflammatory markers including IkappaB kinase β (IKKβ), nuclear factor kappa B (NFκB), tumor necrosis factor α (TNF-α), interleukin-6 (IL-6) and IL-1β, and immune-related proteins including inducible nitric oxide synthase (iNOS), toll-like receptor 2 (TLR2) and cluster of differentiation 14 (CD14) were examined by Western blotting. Results Olanzapine treatments for 1, 8 and 36 days significantly activated the inflammatory IKKβ/NFκB signaling, and increased the expression of TNF-α, IL-6, IL-1β and immune-related proteins such as iNOS, TLR4 and CD14. Olanzapine treatment for 1 day, 8 and 36 days also induced ER stress in the PFC. Co-treatment with an ER stress inhibitor, 4-phenylbutyrate, inhibited olanzapine-induced inflammation and the immune response in the PFC. Conclusion These results suggested olanzapine exposure could be a factor that induces central inflammation and immunological abnormities in schizophrenia subjects. Olanzapine induces PFC inflammation and immune response, possibly via activating ER stress signaling.

Calves born to multiparous Holstein cows fed during the last 30 d of pregnancy 2 different cobalt sources [cobalt glucoheptonate (CoPro) or cobalt pectin (CoPectin)], folic acid (FOA), and rumen-protected methionine (RPM) were used to study neonatal immune responses after ex vivo lipopolysaccharide (LPS) challenge. Groups were (n = 12 calves/group) CoPro, FOA+CoPro, FOA+CoPectin, and FOA+CoPectin+RPM. Calves were weighed at birth and blood collected at birth (before colostrum), 21 d of age, and 42 d of age (at weaning). Growth performance was recorded once a week during the first 6 wk of age. Energy metabolism, inflammation, and antioxidant status were assessed at birth through various plasma biomarkers. Whole blood was challenged with 3 µg/mL of LPS or used for phagocytosis and oxidative burst assays. Target genes evaluated by real-time quantitative PCR in whole blood samples were associated with immune response, antioxidant function, and 1-carbon metabolism. The response in mRNA abundance in LPS challenged versus nonchallenged samples was assessed via δ = LPS challenged − LPS nonchallenged samples. Phagocytosis capacity and oxidative burst activity were measured in neutrophils and monocytes, with data reported as ratio (percentage) of CD14 to CH138A-positive cells. Data including all time points were subjected to ANOVA using PROC MIXED in SAS 9.4 (SAS Institute Inc.), with Treatment, Sex, Age, and Treatment × Age as fixed effects. A 1-way ANOVA was used to determine differences at birth, with Treatment and Sex as fixed effects. Calf birth body weight and other growth parameters did not differ between groups. At birth, plasma haptoglobin concentration was lower in FOA+CoPro compared with CoPro calves. We detected no effect for other plasma biomarkers or immune function due to maternal treatments at birth. Compared with CoPro, in response to LPS challenge, whole blood from FOA+CoPectin and FOA+CoPectin+RPM calves had greater mRNA abundance of intercellular adhesion molecule 1 (ICAM1). No effect for other genes was detectable. Regardless of maternal treatments, sex-specific responses were observed due to greater plasma concentrations of haptoglobin, paraoxonase, total reactive oxygen metabolites, nitrite, and β-carotene in female versus male calves at birth. In contrast, whole blood from male calves had greater mRNA abundance of IRAK1, CADM1, and ITGAM in response to LPS challenge at birth. The longitudinal analysis of d 0, 21, and 42 data revealed greater bactericidal permeability-increasing protein (BPI) mRNA abundance in whole blood from FOA+CoPectin versus FOA+CoPro calves, coupled with greater abundance in FOA+CoPro compared with CoPro calves. Regardless of maternal treatments, most genes related to cytokines and cytokine receptors (IL1B, IL10, TNF, IRAK1, CXCR1), toll-like receptor pathway (TLR4, NFKB1), adhesion and migration (ICAM1, ITGAM), antimicrobial function (MPO), and antioxidant function (GPX1) were downregulated over time. Phagocytosis capacity and oxidative burst activity in both neutrophils and monocytes did not differ due to maternal treatment. Regardless of maternal treatments, we observed an increase in the percentage of neutrophils capable of phagocytosis and oxidative burst activity over time. Overall, these preliminary assessments suggested that maternal supplementation with FOA and Co combined with RPM had effects on a few plasma biomarkers of inflammation at birth and molecular responses associated with inflammatory mechanisms during the neonatal period.

Introduction. At the current stage of the study of atherosclerosis, it has been established that chronic activation of innate immunity, causing persistent low-intensity sterile inflammation, plays a crucial role at all stages of atherogenesis. Laboratory evaluation of signaling pathways associated with molecular patterns (DAMPs) in atherosclerosis and related to cardiovascular diseases (CVD) may contribute to the discovery of new diagnostic and prognostic markers. Objective: to study the relationship between lipid metabolism parameters and CD36 exposure to circulating monocytes in patientwithout established CVD. Material and methods . The study included 42 patients aged 40–64 years without established atherosclerotic CVD, 19 (45.2 %) men and 23 (54.7 %) women. Dyslipidemia was detected in 95.2 % of patients. The blood serum concentrations of total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, glucose, glycated hemoglobin, high-sensitivity C-reactive protein (hs-CRP), creatinine were determined with subsequent calculation of glomerular filtration rate. Phenotyping of circulating monocyte subpopulations was performed by flow cytometry on a Navios 6/2 device (Beckman Coulter, USA). Results and discussion . According to the results of correlation analysis, non-HDL cholesterol levels were inversely correlated with absolute ( r = –0.394; p = 0.013) and relative ( r = –0.432; p = 0.006) content of CD14 + CD16 ++ CD36 + TLR2 + monocytes. LDL cholesterol levels were also inversely correlated with the relative content of CD14 + CD16 + CD36 + TLR2 + monocytes ( r = –0.417; p = 0.018). According to correlation analysis, the level of non-HDL cholesterol inversely correlated with the intensity of CD36 expression on classical ( r = –0.650; p < 0.0001), intermediate ( r = –0.323; p = 0.045) and non classical ( r = –0.480; p = 0.002) monocytes. Also, CD36 expression intensity on classical ( r = –0.449; p = 0.004) and non-classical ( r = –0.382; p = 0.016) monocytes was inversely correlated with remnant cholesterol levels. In addition, increased non-HLA cholesterol levels were associated with decreased TLR2 expression on CD14 + CD16 ++ monocytes ( r = –0.381; p = 0.018). It should be noted that a decrease in CD36 expression on intermediate monocytes was also associated with an increase in hs-CRP ( r = –0.657; p = 0.003). Conclusion . In patients without established atherosclerotic CVD, an increase in cholesterol content of atherogenic lipoprotein fractions was associated with a decrease in the number of CD14 + CD16 ++ and CD14 + CD16 + monocytes co-expressing CD36 and TLR2 as well as with a decrease in CD36 expression on classical, intermediate and non-classical monocytes.

Background Minimally invasive surgery is a field of rapid development. Evidence from randomized controlled trials in visceral surgery however still falls short of attesting unequivocal superiority to laparoscopic procedures over conventional open approaches with regard to postoperative outcome. The aim of this study was to explore the perioperative immune status of patients undergoing hybrid minimally invasive or conventional open pancreatoduodenectomy in a prospective cohort study. Material and methods Subtyping, quantification and functional analysis of circulating immune cells and determination of cytokine-levels in blood samples from patients receiving either hybrid minimally invasive (laPD) or conventional open pancreatoduodenectomy (oPD) was performed. Samples were taken from 29 patients (laPD: n = 14, oPD: n = 15) prior, during and up to six weeks after surgery. Cells were analyzed by flow cytometry, cytokines/chemokines were measured by proximity extension and enzyme-linked immunoassays. Results Open surgery induced higher levels of circulating inflammatory CD14++CD16+ intermediate monocytes. In contrast, hybrid minimally invasive resection was accompanied by increased numbers of circulating regulatory CD4+CD25+CD127low T-cells and by a reduced response of peripheral blood CD3+CD4+ T-cell populations to superantigen stimulation. Yet, rates of postoperative morbidity and infectious complications were similar. Conclusions In summary, the results of this exploratory study may suggest a more balanced postoperative inflammatory response and a better-preserved immune regulation after hybrid minimally invasive pancreatoduodenectomy when compared to open surgery. Whether these results may translate to or be harnessed for improved patient outcome needs to be determined by future studies including larger cohorts and fully laparoscopic or robotic procedures.

Objective Systemic sclerosis (SSc) is characterized by dysregulation of type I interferon (IFN) signaling. CD52 is known for its immunosuppressive functions in T cells. This study was undertaken to investigate the role of CD52 in monocyte adhesion and type I IFN signaling in patients with SSc. Methods Transcriptome profiles of circulating CD14+ monocytes from patients with limited cutaneous SSc (lcSSc), patients with diffuse cutaneous SSc (dcSSs), and healthy controls were analyzed by RNA sequencing. Levels of CD52, CD11b/integrin αΜ, and CD18/integrin β2 in whole blood were assessed by flow cytometry. CD52 expression was analyzed in relation to disease phenotype (early, lcSSc, dcSSc) and autoantibody profiles. The impact of overexpression, knockdown, and antibody blocking of CD52 was analyzed by gene and protein expression assays and functional assays. Results Pathway enrichment analysis indicated an increase in adhesion- and type I IFN-related genes in monocytes from SSc patients. These cells displayed up-regulated expression of CD11b/CD18, reduced expression of CD52, and enhanced adhesion to intercellular adhesion molecule 1 and endothelial cells. Changes in CD52 expression were consistent with the SSc subtypes, as well as with immunosuppressive treatments, autoantibody profiles, and monocyte adhesion properties in patients with SSc. Overexpression of CD52 led to decreased levels of CD18 and monocyte adhesion, while knockdown of CD52 increased monocyte adhesion. Experiments with the humanized anti-CD52 monoclonal antibody alemtuzumab in blood samples from healthy controls increased monocyte adhesion and CD11b/CD18 expression, and enhanced type I IFN responses. Monocytic CD52 expression was up-regulated by interleukin-4 (IL-4)/IL-13 via the STAT6 pathway, and was down-regulated by lipopolysaccharide and IFNs α, β, and γ in a JAK1 and histone deacetylase IIa (HDAC IIa)-dependent manner. Conclusion Down-regulation of the antiadhesion CD52 antigen in CD14+ monocytes represents a novel mechanism in the pathogenesis of SSc. Targeting of the IFN-HDAC-CD52 axis in monocytes might represent a new therapeutic option for patients with early SSc.

Cocaine use disorder (CUD) is a major public health issue associated with physical, social, and psychological problems. Excessive and repeated cocaine use induces oxidative stress leading to a systemic inflammatory response. Cannabidiol (CBD) has gained substantial interest for its anti-inflammatory properties, safety, and tolerability profile. However, CBD anti-inflammatory properties have yet to be confirmed in humans. This exploratory study is based on a single-site randomized controlled trial that enrolled participants with CUD between 18 and 65 years, randomized (1:1) to daily receive either CBD (800 mg) or placebo for 92 days. The trial was divided into a 10-day detoxification (phase I) followed by a 12-week outpatient follow-up (phase II). Blood samples were collected from 48 participants at baseline, day 8, week 4, and week 12 and were analyzed to determine monocytes and lymphocytes phenotypes, and concentrations of various inflammatory markers such as cytokines. We used generalized estimating equations to detect group differences. Participants treated with CBD had lower levels of interleukin-6 (p = 0.017), vascular endothelial growth factor (p = 0.032), intermediate monocytes CD14(+)CD16(+) (p = 0.024), and natural killer CD56(neg)CD16(hi) (p = 0.000) compared with participants receiving placebo. CD25(+)CD4(+)T cells were higher in the CBD group (p = 0.007). No significant group difference was observed for B lymphocytes. This study suggests that CBD may exert anti-inflammatory effects in individuals with CUD.

Background. Metastatic osteosarcoma is a common and fatal bone tumor. Several studies have found that tumor-infiltrating immune cells play pivotal roles in the progression of metastatic osteosarcoma. However, the heterogeneity of infiltrating immune cells across metastatic and primary osteosarcoma remains unclear. Methods. Immune infiltration analysis was carried out via the “ESTIMATE” and “xCell” algorithms in primary and metastatic osteosarcoma. Then, we evaluated the prognostic value of infiltrating immune cells in 85 osteosarcomas through the Kaplan–Meier (K-M) and receiver operating characteristic (ROC) curve. Infiltrations of macrophage M1 and M2 were evaluated in metastatic osteosarcoma, as well as their correlation with immune checkpoints. Macrophage-related prognostic genes were identified through Weighted Gene Coexpression Network Analysis (WGCNA), Lasso analysis, and Random Forest algorithm. Finally, a macrophage-related risk model had been constructed and validated. Results. Macrophages, especially the macrophage M1, sparingly infiltrated in metastatic compared with the primary osteosarcoma and predicted the worse overall survival (OS) and disease-free survival (DFS). Macrophage M1 was positively correlated with immune checkpoints PDCD1, CD274 (PD-L1), PDCD1LG2, CTLA4, and TIGIT. In addition, four macrophage-related prognostic genes (IL10, VAV1, CD14, and CCL2) had been identified, and the macrophage-related risk model had been validated to be reliable for evaluating prognosis in osteosarcoma. Simultaneously, the risk score showed a strong correlation with several immune checkpoints. Conclusion. Macrophages potentially contribute to the regulation of osteosarcoma metastasis. It can be used as a candidate marker for metastatic osteosarcoma’ prognosis and immune checkpoints blockades (ICBs) therapy. We constructed a macrophage-related risk model through machine-learning, which might help us evaluate patients’ prognosis and response to ICBs therapy.

Objective Osteoarthritis (OA) is initiated by pathogenic factors produced by multiple stimuli, including mechanical stress, metabolic stress, and/or inflammaging. This study was undertaken to identify novel low‐grade inflammation–associated pathogenic mediators of OA. Methods Candidate pathogenic molecules were screened using microarray data obtained from chondrocytes exposed to OA‐associated catabolic factors. In mice with OA generated by destabilization of the medial meniscus (DMM), low‐grade inflammation was induced by a high‐fat diet or endotoxemia. Functions of candidate molecules in OA pathogenesis were examined using primary‐culture chondrocytes from mice with DMM‐induced OA, following intraarticular injection of adenovirus expressing the candidate gene. Specific functions of candidate genes were evaluated using whole‐body gene‐knockout mice. Results Bioinformatics analysis identified multiple candidate pathogenic factors that were associated with low‐grade inflammation, including components of the Toll‐like receptor (TLR) signaling pathways (e.g., TLR‐2, TLR‐4, lipopolysaccharide binding protein [LBP], and CD14). Overexpression of the individual TLR signaling components in mouse joint tissue did not alter cartilage homeostasis. However, the low‐grade inflammation induced by a high‐fat diet or endotoxemia markedly enhanced posttraumatic OA cartilage destruction in mice, and this exacerbation of cartilage destruction was significantly abrogated in LBP−/− and CD14−/− mice. Additionally, LBP and CD14 were found to be necessary for the expression of matrix‐degrading enzymes in mouse chondrocytes treated with proinflammatory cytokines. Conclusion LBP and CD14, which are accessory molecules of TLRs, are necessary for the exacerbation of posttraumatic OA cartilage destruction resulting from low‐grade inflammation, such as that triggered by a high‐fat diet or endotoxemia.

Defective phagocytosis has been shown in chronic obstructive pulmonary disease (COPD) bronchoalveolar lavage and blood monocyte‐derived macrophages. Phagocytic capabilities of sputum macrophages and neutrophils in COPD are unknown. We investigated phagocytosis in these cells from COPD patients and controls. Phagocytosis of Streptococcus pneumoniae or fluorescently labelled non‐typeable Haemophilus influenzae (NTHi) by sputum macrophages and neutrophils was determined by gentamycin protection assay (COPD; n = 5) or flow cytometry in 14 COPD patients, 8 healthy smokers (HS) and 9 healthy never‐smokers (HNS). Sputum macrophages and neutrophils were differentiated by adherence for the gentamycin protection assay or receptor expression (CD206 and CD66b, respectively), by flow cytometry. The effects of NTHi on macrophage expression of CD206 and CD14 and neutrophil expression of CD16 were determined by flow cytometry. There was greater uptake of S. pneumoniae [~10‐fold more colony‐forming units (CFU)/ml] by sputum neutrophils compared to macrophages in COPD patients. Flow cytometry showed greater NTHi uptake by neutrophils compared to macrophages in COPD (67 versus 38%, respectively) and HS (61 versus 31%, respectively). NTHi uptake by macrophages was lower in HS (31%, p = 0.019) and COPD patients (38%, p = 0.069) compared to HNS (57%). NTHi uptake by neutrophils was similar between groups. NTHi exposure reduced CD206 and CD14 expression on macrophages and CD16 expression on neutrophils. Sputum neutrophils showed more phagocytic activity than macrophages. There was some evidence that bacterial phagocytosis was impaired in HS sputum macrophages, but no impairment of neutrophils was observed in HS or COPD patients. These results highlight the relative contributions of neutrophils and macrophages to bacterial clearance in COPD., Using flow cytometry analysis we showed greater phagocytic activity of sputum neutrophils compared to sputum macrophages in COPD patients and smoking and non‐smoking controls. While we showed some evidence that bacterial phagocytosis was impaired in sputum macrophages from smokers, there was no impairment of phagocytosis in sputum neutrophils form smokers or COPD patients. Also Haemophilus influenzae exposure reduced CD206 and CD14 expression on macrophages and CD16 expression on neutrophils.

Objective There are limited studies on the association of HIV infection with systemic inflammation during pregnancy. Design A cohort study (N = 220) of pregnant women with HIV (N = 70) (all on antiretroviral therapy) and without HIV (N = 150) were enrolled from an antenatal clinic in Pune, India. Methods The following systemic inflammatory markers were measured in plasma samples using immunoassays: soluble CD163 (sCD163), soluble CD14 (sCD14), intestinal fatty acid-binding protein (I-FABP), C-reactive protein (CRP), alpha 1-acid glycoprotein (AGP), interferon-β (IFNβ), interferon-γ (IFNγ), interleukin (IL)-1β, IL-6, IL-13, IL-17A and tumor necrosis factor α (TNFα). Generalized estimating equation (GEE) and linear regression models were used to assess the association of HIV status with each inflammatory marker during pregnancy and by trimester, respectively. Results Pregnant women with HIV had higher levels of markers for gut barrier dysfunction (I-FABP), monocyte activation (sCD14) and markers of systemic inflammation (IL-6 and TNFα), but surprisingly lower levels of AGP, an acute phase protein, compared to pregnant women without HIV, with some trimester-specific differences. Conclusions Our data show that women with HIV had higher levels of markers of gut barrier dysfunction, monocyte activation and systemic inflammation. These markers, some of which are associated with preterm birth, might help explain the increase in adverse birth outcomes in women with HIV and could suggest targets for potential interventions.

Ken Takata, Kentaro Uchida, Shotaro Takano, Manabu Mukai, Gen Inoue, Hiroyuki Sekiguchi, Jun Aikawa, Masayuki Miyagi, Dai Iwase, Masashi Takaso Department of Orthopedic Surgery, Kitasato University School of Medicine, Sagamihara City, Kanagawa, 252-0374, JapanCorrespondence: Kentaro UchidaDepartment of Orthopedic Surgery, Kitasato University School of Medicine, 1-15-1 Minami-Ku Kitasato, Sagamihara City, Kanagawa, 252-0374, JapanTel/Fax +81-042-778-9217Email kuchida@med.kitasato-u.ac.jpPurpose: Obesity is associated with the risk of developing knee osteoarthritis (KOA). Furthermore, synovial basic fibroblast growth factor (bFGF) is linked to the severity of KOA. We previously demonstrated that bFGF and mast cell (MC) marker expression were elevated in the synovial tissues (ST) of KOA patients with obesity. However, it remains unclear whether MCs contribute to bFGF expression and regulation.Patients and Methods: Radiographically diagnosed KOA patients (n=249) were assigned to groups based on the body mass index (BMI) classifications used by the World Health Organization: normal-weight (NW, BMI < 25 kg/m2, n=95), overweight (OW, BMI ≥ 25 and < 30, n=109) and obese (OB, ≥ 30 kg/m2, n=45). BFGF expression in the ST was examined using quantitative polymerase chain reaction and compared across the BMI groups. Additionally, BFGF and interleukin (IL) 13 expression were examined in freshly extracted MC-rich (THY-1-, CD3-, CD14-, and CD19-) and MC-poor (THY-1+, CD3+, CD14+, or CD19+) fractions from ST. Moreover, regulation of BFGF expression by IL-13 was studied in CD14-negative (fibroblast-rich) and CD14-positive (M&phis;-rich) and cells in culture.Results: BFGF expression was significantly higher in OB than in NW patients. Furthermore, although IL13 was significantly higher in the MC-rich than the MC-poor fraction, BFGF expression was comparable. Recombinant human IL-13 stimulated expression of BFGF in synovial fibroblast cells.Conclusion: BFGF expression is higher in the ST of KOA patients with obesity. Increased numbers of MCs may contribute to the elevated BFGF expression through IL-13 in KOA patients with obesity.Keywords: obesity, mast cell, osteoarthritis, BFGF, IL13, synovial tissues

Algal polysaccharide activates macrophages to alter physiologic biomarkers to drive the immunomodulatory phenotype, but it lacks specific biomarkers involved in the biochemical underpinning process. Here, we undertook an extensive analysis of the RAW 264.7 macrophages induced by an immunostimulating sulfated polysaccharide from Caulerpa racemosa var. peltata (CRVP-1) employing combined transcriptomic, proteomic, and metabolomic analyses to reveal the molecular details occurring in the CRVP-1-induced immunomodulatory process. The omics profiling of CRVP-1-activated macrophage demonstrated a total of 8844 genes (4354 downregulated and 4490 upregulated), 1243 proteins (620 downregulated and 623 upregulated), and 68 metabolites (52 downregulated and16 upregulated). Further, the co-mapped correlation network of omics combined with Western blot and immunofluorescence staining indicated that the cluster of differentiation 14 (CD14) might assist Toll-like receptor 4 (TLR4) involved in nuclear factor kappa-B (NF-κB) signaling pathway to drive the immunomodulatory phenotype. Together, our results discover novel physiologic biomarkers in the immunomodulatory activities of algal polysaccharides.

Background: Inflammation process is an important determinant for subsequent changes in cardiac function and remodeling after acute myocardial infarction (MI). Recent studies have implicated that ALK4 haplodeficiency improves cardiac function after MI. However, it remains unknown if the beneficial effects are partly attributed to ALK4 haplodeficiency-induced modulation on inflammatory response in the inflammatory phase of MI. In this research, we aimed to explore the mechanism of ALK4 haplodeficiency in the inflammatory stage of MI. Methods: ALK4, CD16, and CD14 were detected in peripheral blood mononuclear cells (PBMCs) isolated from MI patients and healthy volunteers. ALK4 haplodeficiency (ALK4+/-) mice and wild-type (WT) littermates were randomly divided into the sham group and the MI group. Inflammation cytokines and chemokines were measured. Echocardiography and intracardiac electrophysiological recordings were performed on the 3rd day and the 7th day after MI operation. ALK4 expression and inflammation cytokines were also detected in LPS- or IL-4-stimulated bone marrow-derived macrophages (BMDM) from the ALK4+/- mice and WT littermates. Results: ALK4 gene expression in circulating monocytes of MI patients was higher than that in those of healthy volunteers. Cardiac inflammation and vulnerability of ventricular arrhythmia after acute myocardial injury are significantly alleviated in ALK4+/- mice as compared to WT littermates. On the 3rd day post-MI, the level of M1 macrophages were decreased in ALK4+/- mice as compared to WT littermates, while the level of M2 macrophages were increased on the 7th day post-MI. BMDM isolated from ALK4+/- mice displayed reduced secretion of pro-inflammation cytokines after stimulation by LPS in hypoxic condition and increased secretion of anti-inflammation cytokines after stimulation by IL-4. As a result, the haplodeficiency of ALK4 might be responsible for reduced inflammation response in the post-MI stage. Conclusions: ALK4 haplodeficiency reduces cardiac inflammation, improves cardiac function, and finally reduces the vulnerability of ventricular arrhythmia in the inflammatory stage after MI.

Blood levels of acute-phase protein α1-acid glycoprotein (AGP, orosmucoid) increase in patients with cancer. Although AGP is produced from hepatocytes following stimulation by immune cell–derived cytokines under conditions of inflammation and tumorigenesis, the functions of AGP in tumorigenesis and tumor progression remain unknown. In the present study, we revealed that AGP contributes directly to tumor development by induction of programmed death ligand 1 (PD-L1) expression and IL6 production in macrophages. Stimulation of AGP induced PD-L1 expression in both human monocyte–derived macrophages through STAT1 activation, whereas AGP had no direct effect on PD-L1 expression in tumor cells. AGP also induced IL6 production from macrophages, which stimulated proliferation in tumor cells by IL6R-mediated activation of STAT3. Furthermore, administration of AGP to AGP KO mice phenocopied effects of tumor-associated macrophages (TAM) on tumor progression. AGP decreased IFNγ secretion from T cells and enhanced STAT3 activation in subcutaneous tumor tissues. In addition, AGP regulated PD-L1 expression and IL6 production in macrophages by binding with CD14, a coreceptor for Toll-like receptor 4 (TLR4), and inducing TLR4 signaling. These results provide the first evidence that AGP is directly involved in tumorigenesis by interacting with TAMs and that AGP might be a target molecule for anticancer therapy. Significance: AGP-mediated suppression of antitumor immunity contributes to tumor progression by inducing PD-L1 expression and IL6 production in TAMs.

Background The expression profiles of the intestinal mucosa have not been comprehensively investigated in asthma. We aimed to explore this in the Correlated Expression and Disease Association Research (CEDAR) patient cohort. Methods Differential expression analysis of ileal, transverse colon, and rectal biopsies were supplemented by a comparison of transcriptomes from platelets and leukocytes subsets, including CD4+, CD8+, CD14+, CD15+, and CD19+ cells. Asthma patients (n = 15) and controls (n = 15) had similar age (p = 0.967), body mass index (p = 0.870), similar numbers of females (80%) and smoking rates (13.3%). Results Significant differential expression was found in the ileum alone, and not in any other cell/tissue types. More genes were found to be overexpressed (1,150) than under-expressed (380). The most overexpressed genes included Fc Fragment of IgG Binding Protein (FCGBP, logFC = 3.01, pFDR = 0.015), Mucin 2 (MUC2, logFC = 2.78, pFDR = 0.015), and Alpha 1B Defensin (DEFA1B, logFC = 2.73, pFDR = 0.024). Gene ontology implicated the immune system, including interleukins 4 and 13, as well as antimicrobial peptides in this overexpression. There was concordance of gene over- (STAT1, XBP1) and underexpression (NELF, RARA) in asthma and Crohn’s disease ileum when our results were compared to another dataset (p = 3.66 × 10–7). Conclusion Ileal mucosa in asthma exhibits a specific transcriptomic profile, which includes the overexpression of innate immune genes, mostly characteristic of Paneth and goblet cells, in addition to other changes that may resemble Crohn’s disease.

Cholangiocarcinoma (CC) is an uncommon malignancy with increasing incidence and dismal prognosis. We conducted a comprehensive analysis of the CC tumor immune microenvironment (TIME) based on tumor location to identify therapeutic targets. We hypothesized that the TIME of CC would vary by primary tumor location and that high tumor infiltration by CD8+ T cells and low infiltration by M2 macrophages would be associated with improved survival. A retrospective analysis was conducted of 99 CC tumor samples surgically resected between 2000 and 2014. Tissue microarrays were constructed from each tumor and stained by immunohistochemistry for 24 markers of immune cells, immune activation or inhibition, programmed cell death-ligand 1, and mesothelin. Most tumors were amply infiltrated with by CD4+, CD8+, and FoxP3+ T cells, as well as by myeloid cells. Mesothelin expression ≥1+ by immunohistochemistry was found in 68% of tumors. We identified higher densities of M1 macrophages in primary distal extrahepatic CC, as well as metastatic lesions. Mesothelin expression was also significantly higher in distal extrahepatic CC. There was no association with survival of infiltration by CD4+, CD8+, or FoxP3+ T cells, mesothelin expression, or programmed cell death-ligand 1 percentage expression, however, high CD14+ myeloid cells and high CD163+ M2 macrophages were associated with worse survival. In conclusion, the CC TIME is a heterogenous milieu highly infiltrated by innate and adaptive immune cells, which differs based on primary tumor location and between primary tumors and metastatic lesions. The correlation of intratumoral M2 macrophages and myeloid cells with a worse prognosis may suggest promising immunotherapeutic targets in CC.