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Neonatal BCG vaccination is associated with a reduction in all-cause mortality. This study reports altered cytokine responses to heterologous pathogens and TLR ligands in BCG-vaccinated neonates compared to non-BCG-vaccinated controls, consistent with modulation of innate immunity. Abstract Background BCG vaccination is associated with a reduction in all-cause infant mortality in high-mortality settings. The underlying mechanisms remain uncertain, but long-term modulation of the innate immune response (trained immunity) may be involved. Methods Whole-blood specimens, collected 7 days after randomization from 212 neonates enrolled in a randomized trial of neonatal BCG vaccination, were stimulated with killed pathogens and Toll-like receptor (TLR) ligands to interrogate cytokine responses. Results BCG-vaccinated infants had increased production of interleukin 6 (IL-6) in unstimulated samples and decreased production of interleukin 1 receptor antagonist, IL-6, and IL-10 and the chemokines macrophage inflammatory protein 1α (MIP-1α), MIP-1β, and monocyte chemoattractant protein 1 (MCP-1) following stimulation with peptidoglycan (TLR2) and R848 (TLR7/8). BCG-vaccinated infants also had decreased MCP-1 responses following stimulation with heterologous pathogens. Sex and maternal BCG vaccination status interacted with neonatal BCG vaccination. Conclusions Neonatal BCG vaccination influences cytokine responses to TLR ligands and heterologous pathogens. This effect is characterized by decreased antiinflammatory cytokine and chemokine responses in the context of higher levels of IL-6 in unstimulated samples. This supports the hypothesis that BCG vaccination modulates the innate immune system. Further research is warranted to determine whether there is an association between these findings and the beneficial nonspecific (heterologous) effects of BCG vaccine on all-cause mortality.

Naturally abundant quinones are important molecules, which play essential roles in various biological processes due to their reduction potential. In contrast to their universality, the investigation of reactions between quinones and proteins remains sparse. Herein, we report the development of a convenient strategy to protein modification via a biomimetic quinone-mediated oxidation at the N -terminus. By exploiting unique reactivity of an ortho- quinone reagent, the α-amine of protein N -terminus is oxidized to generate aldo or keto handle for orthogonal conjugation. The applications have been demonstrated using a range of proteins, including myoglobin, ubiquitin and small ubiquitin-related modifier 2 (SUMO2). The effect of this method is further highlighted via the preparation of a series of 17 macrophage inflammatory protein 1β (MIP-1β) analogs, followed by preliminary anti-HIV activity and cell viability assays, respectively. This method offers an efficient and complementary approach to existing strategies for N -terminal modification of proteins. Methods for selective modification of the N-terminus of proteins are of high interest, but mostly require specific amino acid residues. Here, the authors report a selective and fast method for N-terminal modification of proteins based on quinone-mediated oxidation of the alpha-amine to aldehyde or ketone, and apply it to diverse proteins.

Macrophage inflammatory protein‐1 (MIP‐1), MIP‐1α (CCL3) and MIP‐1β (CCL4) are chemokines crucial for immune responses towards infection and inflammation. Both MIP‐1α and MIP‐1β form high‐molecular‐weight aggregates. Our crystal structures reveal that MIP‐1 aggregation is a polymerization process and human MIP‐1α and MIP‐1β form rod‐shaped, double‐helical polymers. Biophysical analyses and mathematical modelling show that MIP‐1 reversibly forms a polydisperse distribution of rod‐shaped polymers in solution. Polymerization buries receptor‐binding sites of MIP‐1α, thus depolymerization mutations enhance MIP‐1α to arrest monocytes onto activated human endothelium. However, same depolymerization mutations render MIP‐1α ineffective in mouse peritoneal cell recruitment. Mathematical modelling reveals that, for a long‐range chemotaxis of MIP‐1, polymerization could protect MIP‐1 from proteases that selectively degrade monomeric MIP‐1. Insulin‐degrading enzyme (IDE) is identified as such a protease and decreased expression of IDE leads to elevated MIP‐1 levels in microglial cells. Our structural and proteomic studies offer a molecular basis for selective degradation of MIP‐1. The regulated MIP‐1 polymerization and selective inactivation of MIP‐1 monomers by IDE could aid in controlling the MIP‐1 chemotactic gradient for immune surveillance. The two closely related MIP chemokines, CCL3 and CCL4, assemble into high‐order polymers that are protected from degradation. MIP‐1 polymers can dissociate into active MIP‐1 monomers that in turn are selectively degraded by the insulin‐degrading enzyme.

In view of the previously described anti-inflammatory effects of insulin, we investigated the potential suppressive effect of insulin on plasma concentrations and expression of the chemokines, monocyte chemoattractant protein-1 (MCP-1) and regulated on activation normal T-cell expressed and secreted (RANTES) and their receptors, chemokine receptor (CCR)-2 and CCR-5, in mononuclear cells (MNCs). We also investigated the effect of insulin on other chemokines. Ten obese type 2 diabetic patients were infused with insulin (2 units/h with 100 ml of 5% dextrose/h) for 4 h. Another 8 and 6 type 2 diabetic patients were infused with 100 ml of 5% dextrose/h or saline for 4 h, respectively, and served as control subjects. Blood samples were obtained at 0, 2, 4, and 6 h. Insulin infusion significantly suppressed the plasma concentrations of MCP-1, eotaxin, and RANTES and the expression of RANTES, macrophage inflammatory protein (MIP)-1beta, CCR-2, and CCR-5 in MNCs at 2 and 4 h. Dextrose and saline infusions did not alter these indexes. A low-dose infusion of insulin suppresses the plasma concentration of key chemokines, MCP-1, and RANTES, and the expression of their respective receptors, CCR-2 and CCR-5, in MNCs. Insulin also suppresses the expression of RANTES and MIP-1beta in MNCs. These actions probably contribute to the comprehensive anti-inflammatory effect of insulin.

Mesenchymal stem cells (MSCs) are recruited from BM to the stroma of developing tumors, where they serve as critical components of the tumor microenvironment by secreting growth factors, cytokines, and chemokines. The role of MSCs in colorectal cancer (CRC) progression was controversial. In this study, we found that C-C chemokine receptor type 5 (CCR5) ligands (i.e., C-C motif chemokine ligand 3 (CCL3), CCL4, and CCL5) were highly produced from MSCs using a chemokine array screening with conditioned media from the cultured human MSCs. A relatively strong CCR5 expression could be detected within the cytoplasm of several CRC cell lines. Regarding the effect of MSC, we found that the xenografts in which CCR5-overexpressing HCT116 cells were inoculated into immunocompromised mice were highly promoted in vivo by a mixture with MSCs. Notably, the CCR5 inhibitor, maraviroc, significantly abolished the MSC-induced tumor growth in vivo. In human clinical specimens ( n  = 89), 20 cases (29%) were high for CCR5, whereas 69 cases (71%) were low. Statistical analyses indicated that CCR5 expression in primary CRC was associated with CRC patients’ prognosis. Especially, stage III/IV patients with CCR5-high CRCs exhibited a significantly poorer prognosis than those with CCR5-low CRCs. Furthermore, we investigated the effects of preoperative serum CCR5 ligands on patients’ prognosis ( n  = 114), and found that CRC patients with high serum levels of CCL3 and CCL4 exhibited a poorer prognosis compared to those with low levels of CCL3 and CCL4, while there was no association between CCL5 and prognosis. These results suggest that the inhibition of MSC–CRC interaction by a CCR5 inhibitor could provide the possibility of a novel therapeutic strategy for CRC, and that serum levels of CCL3 and CCL4 could be predictive biomarkers for the prognosis of CRC patients.

Lipopolysaccharides (LPS) is well known to manifest a miscarriage-inducing effector during early pregnancy and activate macrophage to induce M1 macrophage polarization. However, the role of macrophage polarization in LPS-related miscarriage-inducing effect is not apparent. In this work, gene expression changes and the percentage of M1/M2 macrophages and monocytes in LPS-induced miscarried uterus were firstly analyzed by RNA sequencing (RNA-seq) and Flow Cytometry. To explore the origin that contributes to M1/M2 macrophage differentiation, the expression of monocyte chemotactic protein (MCP-1), CCL3, and CCL4, chemokines related to monocyte/macrophage migration, was tested by quantitative real time PCR (qRT-PCR). We found that percentage of M1 macrophages rose, while the percentage of M2 macrophages declined down in the injected mice uterus. Meanwhile, the percentage of M1 and M2 macrophages showed no significant difference in the spleens of LPS injected mice compared to PBS injected control mice. Expression of Mcp-1, Ccl3, and Ccl4 and numbers of monocytes were remarkably up-regulated in LPS-induced miscarried mice uterus. These results indicated that polarization and proportion changes of macrophage in the uterus may contribute to miscarriage. Our work provides new evidence correlating the aberrant regulation of M1/M2 macrophage polarization with deleterious miscarriage-inducing effects. This will help us understand the roles of critical immune cell differentiation in maintaining normal pregnancy.

Cytotoxic T lymphocytes (CTLs) are thought to arrive at target sites either via random search or following signals by other leukocytes. Here, we reveal independent emergent behaviour in CTL populations attacking tumour masses. Primary murine CTLs coordinate their migration in a process reminiscent of the swarming observed in neutrophils. CTLs engaging cognate targets accelerate the recruitment of distant T cells through long-range homotypic signalling, in part mediated via the diffusion of chemokines CCL3 and CCL4. Newly arriving CTLs augment the chemotactic signal, further accelerating mass recruitment in a positive feedback loop. Activated effector human T cells and chimeric antigen receptor (CAR) T cells similarly employ intra-population signalling to drive rapid convergence. Thus, CTLs recognising a cognate target can induce a localised mass response by amplifying the direct recruitment of additional T cells independently of other leukocytes. Immune cells known as cytotoxic T lymphocytes, or CTLs for short, move around the body searching for infected or damaged cells that may cause harm. Once these specialised killer cells identify a target, they launch an attack, removing the harmful cell from the body. CTLs can also recognise and eliminate cancer cells, and can be infused into cancer patients as a form of treatment called adoptive cell transfer immunotherapy. Unfortunately, this kind of treatment does not yet work well on solid tumours because the immune cells often do not infiltrate them sufficiently. It is thought that CTLs arrive at their targets either by randomly searching or by following chemicals secreted by other immune cells. However, the methods used to map the movement of these killer cells have made it difficult to determine how populations of CTLs coordinate their behaviour independently of other cells in the immune system. To overcome this barrier, Galeano Niño, Pageon, Tay et al. employed a three-dimensional model known as a tumouroid embedded in a matrix of proteins, which mimics the tissue environment of a real tumour in the laboratory. These models were used to track the movement of CTLs extracted from mice and humans, as well as human T cells engineered to recognise cancer cells. The experiments showed that when a CTL identifies a tumour cell, it releases chemical signals known as chemokines, which attract other CTLs and recruit them to the target site. Further experiments and computer simulations revealed that as the number of CTLs arriving at the target site increases, this amplifies the chemokine signal being secreted, resulting in more and more CTLs being attracted to the tumour. Other human T cells that had been engineered to recognize cancer cells were also found to employ this method of mass recruitment, and collectively ‘swarm’ towards targeted tumours. These findings shed new light on how CTLs work together to attack a target. It is possible that exploiting the mechanism used by CTLs could help improve the efficiency of tumour-targeting immunotherapies. However, further studies are needed to determine whether these findings can be applied to solid tumours in cancer patients.

Objective. To evaluate the long-term effects of a Mediterranean diet (MeDiet) intervention on the plasma concentrations of inflammatory and plaque stability-related molecules in elderly people at high risk for cardiovascular disease. Design and Setting. 66 participants from primary care centers affiliated with the Hospital Clinic of Barcelona were randomized into 3 groups: MeDiet plus extra virgin olive oil (EVOO) or nuts and a low-fat diet (LFD). At baseline and at 3 and 5 years, we evaluated the changes in the plasma concentrations of 24 inflammatory biomarkers related to the different stages of the atherosclerotic process by Luminex®. Results. At 3 and 5 years, both MeDiet groups showed a significant reduction of IL-6, IL-8, MCP-1, and MIP-1β (P<0.05; all) compared to LFD. IL-1β, IL-5, IL-7, IL-12p70, IL-18, TNF-α, IFN-γ, GCSF, GMCSF, and ENA78 (P<0.05; all) only decreased in the MeDiet+EVOO group and E-selectin and sVCAM-1 (P<0.05; both) in the MeDiet+nuts group. Conclusions. Long-term adherence to MeDiet decreases the plasma concentrations of inflammatory biomarkers related to different steps of atheroma plaque development in elderly persons at high cardiovascular risk.

Background: In cirrhosis, a pathological gut microbiome has been linked with immune dysfunction. A pilot study of probiotic Lactobacillus casei Shirota (LcS) in alcoholic cirrhosis demonstrated significant improvement in neutrophil function. This study aimed to evaluate the efficacy of LcS on neutrophil function and significant infection rates in patients with cirrhosis. Methods: 92 cirrhotic patients (Child–Pugh score ≤10) were randomized to receive LcS or placebo, three times daily for six months. Primary end-points were incidence of significant infection and neutrophil function. Secondary end-points were cytokine profile, endotoxin, bacterial DNA positivity, intestinal permeability and quality of life. Results: Rates of infection, decompensation or neutrophil function did not differ between placebo and probiotic groups. LcS significantly reduced plasma monocyte chemotactic protein-1 and, on subgroup analysis, plasma interleukin-1β (alcoholic cirrhosis), interleukin-17a and macrophage inflammatory protein-1β (non-alcoholic cirrhosis), compared with placebo. No significant differences in intestinal permeability, bacterial translocation or metabolomic profile were observed. Conclusion: LcS supplementation in patients with early cirrhosis is safe. Although no significant infections were observed in either group, LcS improved cytokine profile towards an anti-inflammatory phenotype, an effect which appears to be independent of bacterial translocation.

Sepsis is a systemic inflammatory response to infection, accounting for the most common cause of death in intensive care units. Here, we report that peripheral administration of the hypothalamic neuropeptide orexin improves the survival of mice with lipopolysaccharide (LPS) induced endotoxin shock, a well-studied septic shock model. The effect is accompanied by a suppression of excessive cytokine production and an increase of catecholamines and corticosterone. We found that peripherally administered orexin penetrates the blood-brain barrier under endotoxin shock, and that central administration of orexin also suppresses the cytokine production and improves the survival, indicating orexin's direct action in the central nervous system (CNS). Orexin helps restore body temperature and potentiates cardiovascular function in LPS-injected mice. Pleiotropic modulation of inflammatory response by orexin through the CNS may constitute a novel therapeutic approach for septic shock.