SciCombinator

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Concept: Monocyte

167

Macrophage G2A and CD36 lipid receptors are thought to mediate efferocytosis following tissue injury and thereby prevent excessive inflammation which could compromise tissue repair. To test this, we subjected mice lacking G2A or CD36 receptors to bleomycin-induced lung injury and measured efferocytosis, inflammation and fibrosis. Loss of CD36 (but not G2A) delayed clearance of apoptotic alveolar cells (mean 78% increase in apoptotic cells 7 days post-injury), potentiated inflammation (mean 56% increase in lung neutrophils and 75% increase in lung KC levels 7 days post-injury, 51% increase in lung macrophages 14 days post-injury) and reduced lung fibrosis (mean 41% and 29% reduction 14 and 21 days post-injury respectively). Reduced fibrosis in CD36-/- mice was associated with lower levels of pro-fibrotic TH2 cytokines (IL-9, IL-13, IL-4), decreased expression of the M2 macrophage marker Arginase-1 and reduced interstitial myofibroblasts. G2A, on the other hand, was required for optimal clearance of apoptotic neutrophils during zymosan-induced peritoneal inflammation (50.3% increase in apoptotic neutrophils and 30.6% increase in total neutrophils 24 hours following zymosan administration in G2A-/- mice). Thus, CD36 is required for timely removal of apoptotic cells in the context of lung injury and modulates subsequent inflammatory and fibrotic processes relevant to fibrotic lung disease.

Concepts: Immune system, White blood cell, Monocyte, Fibrosis, Cell biology, Macrophage, Apoptosis, Idiopathic pulmonary fibrosis

167

Inflammation is an important component of normal responses to infection and injury. However, chronic activation of the immune system, due to aberrant responses to normal stimuli, can lead to the establishment of a persistent inflammatory state. Such inflammatory conditions are often debilitating, and are associated with a number of important co-morbidities including cardiovascular disease. Resting non-proliferative tissues have distinctive metabolic activities and requirements, which differ considerably from those in infiltrating immune cells, which are undergoing proliferation and differentiation. Immune responses in tissues may therefore be modulated by the relative abundance of substrates in the inflamed site. In turn immune cell activity can feed back and affect metabolic behaviour of the tissues, as most clearly demonstrated in cachexia - the loss of cellular mass driven by tumour necrosis factor-alpha (TNF-α) a key mediator of the inflammatory response. Here we discuss the potential for metabolomic analysis to clarify the interactions between inflammation and metabolic changes underlying many diseases. We suggest that an increased understanding of the interaction between inflammation and cellular metabolism, energy substrate use, tissue breakdown markers, the microbiome and drug metabolites, may provide novel insight into the regulation of inflammatory diseases.

Concepts: Immune system, Inflammation, Monocyte, Metabolism, Enzyme, Asthma, Infection, Eicosanoid

165

BACKGROUND: Leprosy is a contagious and chronic systemic granulomatous disease caused by Mycobacterium leprae. In the pathogenesis of leprosy, granulomas play a key role, however, the mechanisms of the formation and maintenance of M. leprae granulomas are still not clearly understood. METHODS: To better understand the molecular physiology of M. leprae granulomas and the interaction between the bacilli and human host cells, we developed an in vitro model of human granulomas, which mimicked the in vivo granulomas of leprosy. Macrophages were differentiated from human monocytes, and infected with M. leprae, and then cultured with autologous human peripheral blood mononuclear cells (PBMCs). RESULTS: Robust granuloma-like aggregates were obtained only when the M. leprae infected macrophages were co-cultured with PBMCs. Histological examination showed M. leprae within the cytoplasmic center of the multinucleated giant cells, and these bacilli were metabolically active. Macrophages of both M1 and M2 types co-existed in the granuloma like aggregates. There was a strong relationship between the formation of granulomas and changes in the expression levels of cell surface antigens on macrophages, cytokine production and the macrophage polarization. The viability of M. leprae isolated from granulomas indicated that the formation of host cell aggregates benefited the host, but the bacilli also remained metabolically active. CONCLUSIONS: A simple in vitro model of human M. leprae granulomas was established using human monocyte-derived macrophages and PBMCs. This system may be useful to unravel the mechanisms of disease progression, and subsequently develop methods to control leprosy.

Concepts: Immune system, Monocyte, Tuberculosis, Mycobacterium, PBMC, Granuloma, Leprosy, Mycobacterium leprae

164

Recent studies suggest that leukocytes and erythrocytes play a role in coagulation. However, whether leukocytes, erythrocytes and other hematological variables are associated with risk of venous thrombosis is not well known. To study this, we used data from 2473 venous thrombosis patients and 2935 controls. The variables assessed were: total leukocytes, granulocytes, lymphocytes, monocytes, hematocrit, hemoglobin, erythrocytes and red cell indices (mean corpuscular volume, mean hemoglobin volume, mean corpuscular hemoglobin volume and red cell distribution width). We found a strong dose-response relation for higher red cell distribution width and monocytes with risk of venous thrombosis, with odds ratios of 3.1 (95% confidence interval, 2.0-4.8) and 2.8 (95% confidence interval, 1.3-5.8), respectively, after adjustment for age, sex, C-reactive protein, malignancy and co-morbidities. Monocyte count and red cell distribution width were associated with venous thrombosis even within reference ranges. A low monocyte count (< 0.12x109/L) was associated with a lower venous thrombosis risk after full adjustment (odds ratios 0.6; 95% confidence interval, 0.4-0.8). In summary, high red cell distribution width and blood monocytes, two unexpensive and easily obtainable tests were clearly associated with an increased risk of venous thrombosis. Future studies should evaluate the underlying mechanism and the use of these variables in prediction models for first and recurrent thrombosis.

Concepts: Immune system, White blood cell, Monocyte, Blood, Red blood cell, Hematology, Complete blood count, Mean corpuscular volume

160

Interleukin (IL)-17A signaling via Interleukin 17 receptor A (Il17ra) contributes to the inflammatory host response by inducing recruitment of innate immune cells, but also plays a role in homeostatic neutrophilic granulocyte regulation. Monocytes, the other main innate immune cell, have a longer life span and can pursue multiple differentiation pathways towards tissue macrophages. Monocytes are divided into two subpopulations by expression of the Ly6C/Gr1 surface marker in mice. We here investigated the role of Il17ra in monocyte homeostasis and macrophage generation. In Il17ra(-/-) and in mixed bone marrow chimeric wt/Il17ra(-/-) mice, the concentrations of circulating Il17ra(-/-) Gr1(low) monocytes were significantly decreased compared to wt cells. Pulmonary, splenic and resident peritoneal Il17ra(-/-) macrophages were significantly fewer than of wt origin. Bone marrow progenitor and monocyte numbers were equal, but the proportion of Il17ra(-/-) Gr1(low) monocytes was already decreased at bone marrow level. After monocyte depletion, initial Gr1(high) and Gr1(low) monocyte regeneration of Il17ra(-/-) and wt cells was very similar. However, Il17ra(-/-) Gr1(low) counts were not sustained. After labeling with either fluorescent beads or BrdU, Il17ra(-/-) Gr1(high) monocyte transition to Gr1(low) cells was not detectable unlike wt cells. Monocyte recruitment in acute peritonitis, which is known to be largely due to Gr1(high) cell migration, was unaffected in an identical environment. Unilateral ureteral obstruction induces a less acute inflammatory and fibrotic kidney injury. Compared to wt cells in the same environment, Il17ra(-/-) macrophage accumulation in the kidney was decreased. In the absence of Il17ra on all myeloid cells, renal fibrosis was significantly attenuated. Our data show that Il17ra modulates Gr1(low) monocyte counts and suggest defective Gr1(high) to Gr1(low) monocyte transition as an underlying mechanism. Lack of Il17ra altered homeostatic tissue macrophage formation and diminished renal inflammation and fibrosis. Il17ra appears to be a novel modulator of monocyte phenotype and possible therapeutic target in renal fibrosis.

Concepts: Immune system, White blood cell, Monocyte, Bone marrow, Cell biology, Macrophage, Spleen, Neutrophil granulocyte

149

Heat stress results in a multitude of biological and physiological responses which can become lethal if not properly managed. It has been shown that heat stress causes significant adverse effects in both human and animals. Different approaches have been proposed to mitigate the adverse effects caused by heat stress, among which are special diet and probiotics. We characterized the effect of the yeast fermentate EpiCor (EH) on the prevention of heat stress-related complications in rats. We found that increasing the body temperature of animals from 37.1±0.2 to 40.6±0.2°C by exposure to heat (45°C for 25min) resulted in significant morphological changes in the intestine. Villi height and total mucosal thickness decreased in heat-stressed rats pre-treated with PBS in comparison with control animals not exposed to the heat. Oral treatment of rats with EH before heat stress prevented the traumatic effects of heat on the intestine. Changes in intestinal morphology of heat-stressed rats, pre-treated with PBS resulted in significant elevation of lipopolysaccharides (LPS) level in the serum of these animals. Pre-treatment with EH was effective in the prevention of LPS release into the bloodstream of heat-stressed rats. Our study revealed that elevation of body temperature also resulted in a significant increase of the concentration of vesicles released by erythrocytes in rats, pre-treated with PBS. This is an indication of a pathological impact of heat on the erythrocyte structure. Treatment of rats with EH completely protected their erythrocytes from this heat-induced pathology. Finally, exposure to heat stress conditions resulted in a significant increase of white blood cells in rats. In the group of animals pre-treated with EH before heat stress, the white blood cell count remained the same as in non-heated controls. These results showed the protective effect of the EH product in the prevention of complications, caused by heat stress.

Concepts: Immune system, White blood cell, Monocyte, Lymphocyte, Blood, Red blood cell, Physiology, Bone marrow

143

The heterotrimeric globular head (gC1q) domain of human C1q is made up of the C-terminal ends of the three individual chains, ghA, ghB, and ghC. A candidate receptor for the gC1q domain is a multi-functional pattern recognition protein, gC1qR. Since understanding of gC1qR and gC1q interaction could provide an insight into the pleiotropic functions of gC1qR, this study was undertaken to identify the gC1qR-binding site on the gC1q domain, using the recombinant ghA, ghB, and ghC modules and their substitution mutants. Our results show that ghA, ghB, and ghC modules can interact with gC1qR independently, thus reinforcing the notion of modularity within the gC1q domain of human C1q. Mutational analysis revealed that while Arg162 in the ghA module is central to interaction between gC1qR and C1q, a single amino acid substitution (arginine to glutamate) in residue 114 of the ghB module resulted in enhanced binding. Expression of gC1qR and C1q in adherent monocytes with or without pro-inflammatory stimuli was also analyzed by qPCR; it showed an autocrine/paracrine basis of C1q and gC1qR interaction. Microscopic studies revealed that C1q and gC1qR are colocalized on PBMCs. Cell proliferation assays indicated that ghA, ghB, and ghC modules were able to attenuate phytohemagglutinin-stimulated proliferation of PBMCs. Addition of gC1qR had an additive effect on the anti-proliferative effect of globular head modules. In summary, our results identify residues involved in C1q-gC1qR interaction and explain, to a certain level, their involvement on the immune cell surface, which is relevant for C1q-induced functions including inflammation, infection, and immunity.

Concepts: Immune system, Monocyte, Antibody, Protein, Bacteria, Amino acid, Signal transduction, Immunology

131

Atherosclerosis is a chronic inflammatory disease, and developing therapies to promote its regression is an important clinical goal. We previously established that atherosclerosis regression is characterized by an overall decrease in plaque macrophages and enrichment in markers of alternatively activated M2 macrophages. We have now investigated the origin and functional requirement for M2 macrophages in regression in normolipidemic mice that received transplants of atherosclerotic aortic segments. We compared plaque regression in WT normolipidemic recipients and those deficient in chemokine receptors necessary to recruit inflammatory Ly6Chi (Ccr2-/- or Cx3cr1-/-) or patrolling Ly6Clo (Ccr5-/-) monocytes. Atherosclerotic plaques transplanted into WT or Ccr5-/- recipients showed reduced macrophage content and increased M2 markers consistent with plaque regression, whereas plaques transplanted into Ccr2-/- or Cx3cr1-/- recipients lacked this regression signature. The requirement of recipient Ly6Chi monocyte recruitment was confirmed in cell trafficking studies. Fate-mapping and single-cell RNA sequencing studies also showed that M2-like macrophages were derived from newly recruited monocytes. Furthermore, we used recipient mice deficient in STAT6 to demonstrate a requirement for this critical component of M2 polarization in atherosclerosis regression. Collectively, these results suggest that continued recruitment of Ly6Chi inflammatory monocytes and their STAT6-dependent polarization to the M2 state are required for resolution of atherosclerotic inflammation and plaque regression.

Concepts: Immune system, Inflammation, White blood cell, Monocyte, Atherosclerosis, Macrophage, Blood vessel, Endothelium

93

Food-grade titanium dioxide (TiO2) containing a nanoscale particle fraction (TiO2-NPs) is approved as a white pigment (E171 in Europe) in common foodstuffs, including confectionary. There are growing concerns that daily oral TiO2-NP intake is associated with an increased risk of chronic intestinal inflammation and carcinogenesis. In rats orally exposed for one week to E171 at human relevant levels, titanium was detected in the immune cells of Peyer’s patches (PP) as observed with the TiO2-NP model NM-105. Dendritic cell frequency increased in PP regardless of the TiO2 treatment, while regulatory T cells involved in dampening inflammatory responses decreased with E171 only, an effect still observed after 100 days of treatment. In all TiO2-treated rats, stimulation of immune cells isolated from PP showed a decrease in Thelper (Th)-1 IFN-γ secretion, while splenic Th1/Th17 inflammatory responses sharply increased. E171 or NM-105 for one week did not initiate intestinal inflammation, while a 100-day E171 treatment promoted colon microinflammation and initiated preneoplastic lesions while also fostering the growth of aberrant crypt foci in a chemically induced carcinogenesis model. These data should be considered for risk assessments of the susceptibility to Th17-driven autoimmune diseases and to colorectal cancer in humans exposed to TiO2 from dietary sources.

Concepts: Immune system, Inflammation, Monocyte, Titanium dioxide, Titanium, Rutile, Pigment, Titanium tetrachloride

81

Immune cells in the tumor microenvironment modulate cancer progression and are attractive therapeutic targets. Macrophages and T cells are key components of the microenvironment, yet their phenotypes and relationships in this ecosystem and to clinical outcomes are ill defined. We used mass cytometry with extensive antibody panels to perform in-depth immune profiling of samples from 73 clear cell renal cell carcinoma (ccRCC) patients and five healthy controls. In 3.5 million measured cells, we identified 17 tumor-associated macrophage phenotypes, 22 T cell phenotypes, and a distinct immune composition correlated with progression-free survival, thereby presenting an in-depth human atlas of the immune tumor microenvironment in this disease. This study revealed potential biomarkers and targets for immunotherapy development and validated tools that can be used for immune profiling of other tumor types.

Concepts: Immune system, White blood cell, Monocyte, Antibody, Medicine, Cancer, Macrophage, Immunology