Identifying important parameters in the inflammatory process with a mathematical model of immune cell influx and macrophage polarization.

In an inflammatory setting, macrophages can be polarized to an inflammatory M1 phenotype or to an anti-inflammatory M2 phenotype, as well as existing on a spectrum between these two extremes. Dysfunction of this phenotypic switch can result in a population imbalance that leads to chronic wounds or disease due to unresolved inflammation. Therapeutic interventions that target macrophages have therefore been proposed and implemented in diseases that feature chronic inflammation such as diabetes mellitus and atherosclerosis. We have developed a model for the sequential influx of immune cells in the peritoneal cavity in response to a bacterial stimulus that includes macrophage polarization, with the simplifying assumption that macrophages can be classified as M1 or M2. With this model, we were able to reproduce the expected timing of sequential influx of immune cells and mediators in a general inflammatory setting. We then fit this model to in vivo experimental data obtained from a mouse peritonitis model of inflammation, which is widely used to evaluate endogenous processes in response to an inflammatory stimulus. Model robustness is explored with local structural and practical identifiability of the proposed model a posteriori. Additionally, we perform sensitivity analysis that identifies the population of apoptotic neutrophils as a key driver of the inflammatory process. Finally, we simulate a selection of proposed therapies including points of intervention in the case of delayed neutrophil apoptosis, which our model predicts will result in a sustained inflammatory response. Our model can therefore provide hypothesis testing for therapeutic interventions that target macrophage phenotype and predict outcomes to be validated by subsequent experimentation.

Short-term high-fat diet affects macrophages inflammatory response, early signs of a long-term problem

Obesity is a chronic inflammatory disease that affects millions of people worldwide. Most studies observe the effects of a high-fat diet (HFD) in 10-12 weeks. This work investigated the effects induced by a HFD administered for 6 weeks on the nutritional status of mice and some aspects of the inflammatory response in mouse peritoneal macrophages. Male Swiss Webster mice, 2-3 months of age, were fed a control diet or HFD for 6 weeks. After this period, the mice were euthanized, and peritoneal macrophages were collected for immunoassays and assessment of biochemical parameters. A HFD was associated with increased cholesterol, insulin resistance, C-reactive protein (CRP), leptin, and serum resistin levels. Lipopolysaccharide (LPS)- stimulated adipocyte cultures of animals subjected to a HFD showed increased production of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6). However, peritoneal macrophages of the HFD group showed no changes in the levels of these cytokines. LPS-stimulated peritoneal macrophages from HFD-treated animals showed a reduction in mRNA expression of TNF-α and IL-6, as well as a decrease in expression of the transcription factor nuclear factor-kappa B (NF-kB). In conclusion, HFD treatment for 6 weeks induces similar signs to metabolic syndrome and decreases the capacity of peritoneal macrophages to develop an appropriate inflammatory response to a bacterial component

Changes in the function and phenotype of resident peritoneal macrophages after housing in an enriched environment.

Exposure to an enriched environment (EE) affects not only brain functions but also immune responses upon viral or bacterial infections. In this study, we examined changes in the phagocytic response and chemokine production of resident peritoneal macrophages after mice had been housed under EE conditions for 6 or 8 weeks, and then explored the possibility that EE could cause a change in the macrophage phenotype by means of flow cytometry as well as quantitative RT-PCR. The percentages of EE macrophages phagocytosing S. aureus and apoptotic neutrophils were significantly larger than those of standard environment (SE) macrophages. After coculturing with S. aureus, EE macrophages tended to produce greater amounts of chemokines such as MIP-2, KC and MCP-1 than SE ones, although the increases for MIP-2 and KC were not statistically significant. As compared with SE macrophages, EE macrophages included more CD40-positive cells (M1 marker), and expressed more mRNAs of IL-6 (M1 marker) and IRF4 (M2 marker), and less mRNA of CD38 (M1 marker), suggesting either the possibility that EE macrophages are a mixed population of M1 and M2 macrophages or the possibility that they are a unique population with a mixed M1 and M2 macrophage phenotype.

An M2 Rather than a TH2 Response Contributes to Better Protection against Latency Reactivation following Ocular Infection of Naive Mice with a Recombinant Herpes Simplex Virus 1 Expressing Murine Interleukin-4.

We found previously that altering macrophage polarization toward M2 responses by injection of colony-stimulating factor 1 (CSF-1) was more effective in reducing both primary and latent infections in mice ocularly infected with herpes simplex virus 1 (HSV-1) than M1 polarization by gamma interferon (IFN-γ) injection. Cytokines can coordinately regulate macrophage and T helper (TH) responses, with interleukin-4 (IL-4) inducing type 2 TH (TH2) as well as M2 responses and IFN-γ inducing TH1 as well as M1 responses. We have now differentiated the contributions of these immune compartments to protection against latency reactivation and corneal scarring by comparing the effects of infection with recombinant HSV-1 in which the latency-associated transcript (LAT) gene was replaced with either the IL-4 (HSV-IL-4) or IFN-γ (HSV-IFN-γ) gene using infection with the parental (LAT-negative) virus as a control. Analysis of peritoneal macrophages in vitro established that the replacement of LAT with the IL-4 or IFN-γ gene did not affect virus infectivity and promoted polarization appropriately. Protection against corneal scarring was significantly higher in mice ocularly infected with HSV-IL-4 than in those infected with HSV-IFN-γ or parental virus. Levels of primary virus replication in the eyes and trigeminal ganglia (TG) were similar in the three groups of mice, but the numbers of gC+ cells were lower on day 5 postinfection in the eyes of HSV-IL-4-infected mice than in those infected with HSV-IFN-γ or parental virus. Latency and explant reactivation were lower in both HSV-IL-4- and HSV-IFN-γ-infected mice than in those infected with parental virus, with the lowest level of latency being associated with HSV-IL-4 infection. Higher latency correlated with higher levels of CD8, PD-1, and IFN-γ mRNA, while reduced latency and T-cell exhaustion correlated with lower gC+ expression in the TG. Depletion of macrophages increased the levels of latency in all ocularly infected mice compared with their undepleted counterparts, with macrophage depletion increasing latency in the HSV-IL-4 group greater than 3,000-fold. Our results suggest that shifting the innate macrophage immune responses toward M2, rather than M1, responses in HSV-1 infection would improve protection against establishment of latency, reactivation, and eye disease.IMPORTANCE Ocular HSV-1 infections are among the most frequent serious viral eye infections in the United States and a major cause of virus-induced blindness. As establishment of a latent infection in the trigeminal ganglia results in recurrent infection and is associated with corneal scarring, prevention of latency reactivation is a major therapeutic goal. It is well established that absence of latency-associated transcripts (LATs) reduces latency reactivation. Here we demonstrate that recombinant HSV-1 expressing IL-4 (an inducer of TH2/M2 responses) or IFN-γ (an inducer of TH1/M1 responses) in place of LAT further reduced latency, with HSV-IL-4 showing the highest overall protective efficacy. In naive mice, this higher protective efficacy was mediated by innate rather than adaptive immune responses. Although both M1 and M2 macrophage responses were protective, shifting macrophages toward an M2 response through expression of IL-4 was more effective in curtailing ocular HSV-1 latency reactivation.

Minimal asbestos exposure in germline BAP1 heterozygous mice is associated with deregulated inflammatory response and increased risk of mesothelioma.

Germline BAP1 mutations predispose to several cancers, in particular malignant mesothelioma. Mesothelioma is an aggressive malignancy generally associated with professional exposure to asbestos. However, to date, we found that none of the mesothelioma patients carrying germline BAP1 mutations were professionally exposed to asbestos. We hypothesized that germline BAP1 mutations might influence the asbestos-induced inflammatory response that is linked to asbestos carcinogenesis, thereby increasing the risk of developing mesothelioma after minimal exposure. Using a BAP1(+/-) mouse model, we found that, compared with their wild-type littermates, BAP1(+/-) mice exposed to low-dose asbestos fibers showed significant alterations of the peritoneal inflammatory response, including significantly higher levels of pro-tumorigenic alternatively polarized M2 macrophages, and lower levels of several chemokines and cytokines. Consistent with these data, BAP1(+/-) mice had a significantly higher incidence of mesothelioma after exposure to very low doses of asbestos, doses that rarely induced mesothelioma in wild-type mice. Our findings suggest that minimal exposure to carcinogenic fibers may significantly increase the risk of malignant mesothelioma in genetically predisposed individuals carrying germline BAP1 mutations, possibly via alterations of the inflammatory response.

The role of peritoneal alternatively activated macrophages in the process of peritoneal fibrosis related to peritoneal dialysis.

It has been confirmed that alternatively activated macrophages (M2) participate in tissue remodeling and fibrosis occurrence, but the effect of M2 on peritoneal fibrosis related to peritoneal dialysis (PD) hasn't been elucidated. This study was therefore conducted to assess the association between M2 and peritoneal fibrosis related to PD. In this study, peritoneal fibrosis was induced by intraperitoneal (i.p.) injection of Lactate-4.25% dialysate (100 mL/kg) to C57BL/6J mice for 28 days, and liposome-encapsulated clodronate (LC, the specific scavenger of macrophages) was used to treat the peritoneal fibrosis mice model by i.p. injection at day 18 and day 21. All animals were sacrificed at day 29. Parietal peritonea were stained with Masson's trichrome, and the expression of type I collagen (Col-I), fibronectin, mannose receptor (CD206), transforming growth factor beta (TGF-ß), chemokine receptor 7 (CCR7), chitinase 3-like 3 (Ym-1) and arginase-1 (Arg-1) was determined by Western blotting, immunofluorescence and quantitative real-time PCR. Our results revealed that peritoneal thickness, Col-I, fibronectin, CD206, TGF-ß, Ym-1 and Arg-1 were upregulated in the peritoneal fibrosis mice model, and all of these indexes were downregulated in those treated with LC. Additionally, there was no difference in the level of CCR7 between the model and treatment group. Our study indicated that peritoneal M2 played an important role in the process of peritoneal fibrosis related to PD and might be a potential target for intervention therapy of peritoneal fibrosis.

Aging is associated with an increase in T cells and inflammatory macrophages in visceral adipose tissue.

Age-related adiposity has been linked to chronic inflammatory diseases in late life. To date, the studies on adipose tissue leukocytes and aging have not taken into account the heterogeneity of adipose tissue macrophages (ATMs), nor have they examined how age impacts other leukocytes such as T cells in fat. Therefore, we have performed a detailed examination of ATM subtypes in young and old mice using state of the art techniques. Our results demonstrate qualitative changes in ATMs with aging that generate a decrease in resident type 2 (M2) ATMs. The profile of ATMs in old fat shifts toward a proinflammatory environment with increased numbers of CD206(-)CD11c(-) (double-negative) ATMs. The mechanism of this aging-induced shift in the phenotypic profile of ATMs was found to be related to a decrease in peroxisome proliferator-activated receptor-γ expression in ATMs and alterations in chemokine/chemokine receptor expression profiles. Furthermore, we have revealed a profound and unexpected expansion of adipose tissue T cells in visceral fat with aging that includes a significant induction of regulatory T cells in fat. Our findings demonstrate a unique inflammatory cell signature in the physiologic context of aging adipose tissue that differs from those induced in setting of diet-induced obesity.

Peritoneal macrophages are distinct from monocytes and adherent macrophages.

OBJECTIVE: Peritoneal macrophages are used in many studies related to atherosclerosis. In situ, they are non-adherent and upon culturing, they adhere and function as scavengers of modified lipoproteins and dead apoptotic cells. They also produce growth factors, suggesting that they may provide life-supporting function as well. In this study, we propose that macrophage adherence plays a major role in their function and propose a novel concept that non-adherent macrophages are poor scavengers and may delay the process of apoptosis by secretion of growth factors. METHODS AND RESULTS: We analyzed non-adherent and adherent macrophages for changes in receptor expression, growth factor production and function by microarrays, real-time PCR, and western blot analyses. Our results indicate that adherent macrophages have increased expression of scavenger receptors as compared to fresh peritoneal cells. While genes for many growth factors were expressed in both non-adherent and adherent macrophages, the milk fat globule-epidermal growth factor 8 protein (MFG-E8) that recognizes and takes up apoptotic cells was specifically enhanced in non-adherent cells. Furthermore, early apoptotic endothelial cells demonstrated signs of delayed apoptosis when incubated in the presence of peritoneal lavage fluid that was shown to contain MFG-E8. Functional arrays indicated that peritoneal non-adherent macrophages represent a class of macrophages, distinct from either blood monocytes or adherent cultured macrophages. CONCLUSIONS: These results suggest that the adherence status of macrophages may play a major role in their functions.

[Functional activity of peritoneal macrophages in acute destructive pancreatitis: evaluation, implication].

According to the etiology of acute pancreatitis, the authors identified the most common phenotypes of superficial antigens of peritoneal macrophages: CD14(+) CD25(-)CD64(-)HLA-DR(-) in acute biliary pancreatitis, CD14(+) CD25(+)CD64(+)HLA-DR(+) in nonbiliary pancreatitis; moreover, all the patients were found to have increased expression of early (CD25+) and late (HLA-DR+) markers of cell activation. A comparative study was conducted and the most informative tools to evaluate the adhesive, cytokine-producing, phagocytic, and oxygen-dependent activities of peritoneal macrophages were recognized.

Phenotypic and functional analysis of murine resident and induced peritoneal macrophages.

Primary macrophages from the peritoneal cavities of mice are commonly used ex vivo to produce inflammatory cytokines and test anti-inflammatory agents. Although approximately 1 million peritoneal macrophages can be obtained from an untreated mouse, more than twice that number can be collected 48 to 72 h after intraperitoneal injection of sterile inducing agents such as Brewer thioglycollate broth, casein, and proteose peptone. However, whether 'induced' macrophages are functionally equivalent to 'resident' peritoneal macrophages has been unclear. Flow cytometric analysis revealed significant phenotypic differences between these 2 macrophage types. Resident and induced peritoneal macrophages also demonstrated markedly different capacities to produce the inflammatory cytokines interleukins 6 and 1beta in response to lipopolysaccharide stimulation in vitro. Increased understanding of the differences between resident and induced peritoneal macrophages likely will help investigators decide which macrophage type is appropriate for their in vitro assay needs.

Differential expression of VLA beta 1 (CD29) on monocytes from patients with endometriosis.

PROBLEM: Previous studies have established that in vitro proliferation of endometrial cells is enhanced by peripheral blood monocytes (PBM) and suppressed by peritoneal macrophages (PM) from patients with endometriosis but only suppressed by PBM and PM obtained from normal subjects. The functional activity of PBM and PM is influenced by the engagement of numerous cell surface receptors with their respective physiological ligands. METHOD: In this study, PBM and PM from fertile women (Group 1), women with unexplained infertility (Group 2), and women with limited (Group 3) or severe (Group 4) endometriosis were isolated in order to analyze these cells for the expression of CD54, CD58 and HLA-DR (immunoglobulin supergene antigens) CD18 and CD29 (integrins) and CD44 (an addresin). These cell surface antigens are involved in monocyte/macrophage trafficking, activation, signal transduction and/or adhesion. RESULTS: No differences were detected in the percentage of PBM expressing CD18, CD44, CD54, CD58, or HLA-DR among the four groups of subjects. Furthermore, the density of these antigens expressed on PBM was identical in patients and control subjects. In contrast, the percentage of PBM expressing CD29 (also known as VLA beta 1) and the density of CD29 expressed per cell were significantly reduced (P < 0.01) in patients with limited endometriosis compared to controls and patients with severe disease. Interestingly, although the percentage of CD29+ PBM from women with severe endometriosis was not statistically different from the percentage of CD29+ PBM from controls, the density of CD29 expressed per cell was significantly elevated among patients with severe disease. Analysis of PM from the four subject groups revealed no differences in CD29 expression or density. However, the percentage of PM expressing CD18 was significantly decreased in patients with limited (but not severe) endometriosis. CONCLUSION: Since both CD18 and CD29 play a role in cell trafficking and/or adhesion, alterations in their expression among patients with endometriosis suggest that these integrin beta chains may play a role in the pathogenesis of the disease.