Proliferation Of Resident Macrophages Research Articles

The Intersection of Immune Responses, Microbiota and Pathogenesis in Giardiasis

Abstract Infection with the protozoan parasite, Giardia, can produce a range of symptoms including diarrhea, severe cramps, and nutrient malabsorption. Our lab and others have previously indicated that immune responses can contribute to pathogenesis. It has also been reported that intestinal microbiota can affect the ability of the parasite to colonize laboratory mice. Our goal was to determine if intestinal immune cells were activated following infection and how the microbiota interacted with intestinal immune responses. Wild-type or CCR2 deficient mice were treated with a cocktail of antibiotics and infected with the GS strain. Control mice were not treated with antibiotics and/or were not infected. Changes in the intestinal microbiome and immune responses were determined using 16S sequencing and flow cytometry of isolated lamina propria and intraepithelial lymphocyte populations, respectively. Sucrase activity was also measured in mucosal lysates. Mice treated with antibiotics or infected with Giardia exhibited shifts in their intestinal microbiomes. With or without antibiotics, we observed an increase in the frequency of CD4+ T cells in the lamina propria at day 7 post-infection. We also observed an increase in macrophage populations and activated CD8+ T cells, but only in mice not treated with antibiotics. Macrophage populations increased in wild-type and CCR2-deficient mice, and EdU labeling suggested in situ proliferation of resident macrophages. Finally, sucrase deficiency correlated with activation of CD8+ T cells. Together these data indicate that the intestinal microbiota contribute to immune cell activation during infections and suggest that the microbiota may contribute to different infection outcomes.

Local proliferation initiates macrophage accumulation in adipose tissue during obesity.

Obesity-associated chronic inflammation is characterized by an accumulation of adipose tissue macrophages (ATMs). It is generally believed that those macrophages are derived from peripheral blood monocytes. However, recent studies suggest that local proliferation of macrophages is responsible for ATM accumulation. In the present study, we revealed that both migration and proliferation contribute to ATM accumulation during obesity development. We show that there is a significant increase in ATMs at the early stage of obesity, which is largely due to an enhanced in situ macrophage proliferation. This result was obtained by employing fat-shielded irradiation and bone marrow reconstitution. Additionally, the production of CCL2, a pivotal chemoattractant of monocytes, was not found to be increased at this stage, corroborating with a critical role of proliferation. Nonetheless, as obesity proceeds, the role of monocyte migration into adipose tissue becomes more significant and those new immigrants further proliferate locally. These proliferating ATMs mainly reside in crown-like structures formed by macrophages surrounding dead adipocytes. We further showed that IL-4/STAT6 is a driving force for ATM proliferation. Therefore, we demonstrated that local proliferation of resident macrophages contributes to ATM accumulation during obesity development and has a key role in obesity-associated inflammation.

CSF1 Restores Innate Immunity After Liver Injury in Mice and Serum Levels Indicate Outcomes of Patients With Acute Liver Failure

Background & AimsLiver regeneration requires functional liver macrophages, which provide an immune barrier that is compromised after liver injury. The numbers of liver macrophages are controlled by macrophage colony-stimulating factor (CSF1). We examined the prognostic significance of the serum level of CSF1 in patients with acute liver injury and studied its effects in mice.MethodsWe measured levels of CSF1 in serum samples collected from 55 patients who underwent partial hepatectomy at the Royal Infirmary Edinburgh between December 2012 and October 2013, as well as from 78 patients with acetaminophen-induced acute liver failure admitted to the Royal Infirmary Edinburgh or the University of Kansas Medical Centre. We studied the effects of increased levels of CSF1 in uninjured mice that express wild-type CSF1 receptor or a constitutive or inducible CSF1-receptor reporter, as well as in chemokine receptor 2 (Ccr2)-/- mice; we performed fate-tracing experiments using bone marrow chimeras. We administered CSF1-Fc (fragment, crystallizable) to mice after partial hepatectomy and acetaminophen intoxication, and measured regenerative parameters and innate immunity by clearance of fluorescent microbeads and bacterial particles.ResultsSerum levels of CSF1 increased in patients undergoing liver surgery in proportion to the extent of liver resected. In patients with acetaminophen-induced acute liver failure, a low serum level of CSF1 was associated with increased mortality. In mice, administration of CSF1-Fc promoted hepatic macrophage accumulation via proliferation of resident macrophages and recruitment of monocytes. CSF1-Fc also promoted transdifferentiation of infiltrating monocytes into cells with a hepatic macrophage phenotype. CSF1-Fc increased innate immunity in mice after partial hepatectomy or acetaminophen-induced injury, with resident hepatic macrophage as the main effector cells.ConclusionsSerum CSF1 appears to be a prognostic marker for patients with acute liver injury. CSF1 might be developed as a therapeutic agent to restore innate immune function after liver injury.

Ly6C(high) monocytes become alternatively activated macrophages in schistosome granulomas with help from CD4+ cells.

Alternatively activated macrophages (AAM) that accumulate during chronic T helper 2 inflammatory conditions may arise through proliferation of resident macrophages or recruitment of monocyte-derived cells. Liver granulomas that form around eggs of the helminth parasite Schistosoma mansoni require AAM to limit tissue damage. Here, we characterized monocyte and macrophage dynamics in the livers of infected CX3CR1GFP/+ mice. CX3CR1-GFP+ monocytes and macrophages accumulated around eggs and in granulomas during infection and upregulated PD-L2 expression, indicating differentiation into AAM. Intravital imaging of CX3CR1-GFP+ Ly6Clow monocytes revealed alterations in patrolling behavior including arrest around eggs that were not encased in granulomas. Differential labeling of CX3CR1-GFP+ cells in the blood and the tissue showed CD4+ T cell dependent accumulation of PD-L2+ CX3CR1-GFP+ AAM in the tissues as granulomas form. By adoptive transfer of Ly6Chigh and Ly6Clow monocytes into infected mice, we found that AAM originate primarily from transferred Ly6Chigh monocytes, but that these cells may transition through a Ly6Clow state and adopt patrolling behavior in the vasculature. Thus, during chronic helminth infection AAM can arise from recruited Ly6Chigh monocytes via help from CD4+ T cells.

Establishment of Self-Renewable GM-CSF-Dependent Immature Macrophages In Vitro from Murine Bone Marrow

Macrophages play a key role in the innate immune system. Macrophages are thought to originate from hematopoietic precursors or the yolk sac. Here, we describe the in vitro establishment of self-renewable GM-CSF-dependent immature macrophages (GM-IMs) from murine bone marrow (BM). GM-IMs grow continuously in vitro in conditioned medium containing GM-CSF. The immunophenotype of GM-IMs is F4/80high CD11bhigh CD11clow Ly6Clow. By comparing gene expression in GM-IMs and BM dendritic cells, we found that GM-IMs expressed lower levels of chemokines, cytokines and their receptors. GM-IMs are round in shape, attach loosely to non-coated culture dishes and have a marked phagocytic capacity. These results indicate that GM-IMs are macrophage precursor cells. Following stimulation with LPS, monocyte-like GM-IMs converted to flat macrophage-like cells that tightly adhered to non-coated culture dishes and produced pro-inflammatory cytokines TNFα, IL-6 and IL-1β. These results indicated that GM-IMs differentiated to M1 pro-inflammatory macrophages. This was confirmed by stimulation of GM-IMs with IFNγ, an inducer of M1 markers. GM-IMs showed enhanced expression of M2 macrophage markers such as Arg1 and Retnla following stimulation by Th2 cytokines IL-4 and IL-13. When GM-IMs were injected into mice at sites of wounding, wound repair was enhanced. These results indicate that GM-IMs can differentiate to M2 macrophages. When GM-IMs were injected into clodronate-treated mice, they induced resident macrophage proliferation by producing M-CSF. In conclusion we have established self-renewable GM-CSF-dependent immature macrophages in vitro from murine BM, which differentiate to M1 or M2 macrophages.

Attenuation of MCP-1/CCL2 expression ameliorates neuropathy in a mouse model for Charcot–Marie–Tooth 1X

The chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2) has been previously shown to be an important mediator of macrophage-related neural damage in models of two distinct inherited neuropathies, Charcot-Marie-Tooth (CMT) 1A and 1B. In mice deficient in the gap junction protein connexin 32 (Cx32def), an established model for the X-chromosome-linked dominant form of CMT (CMT1X), we investigated the role of the chemokine in macrophage immigration and neural damage by crossbreeding the Cx32def mice with MCP-1 knockout mutants. In Cx32def mutants typically expressing increased levels of MCP-1, macrophage numbers were strongly elevated, caused by an MCP-1-mediated influx of haematogenous macrophages. Curiously, the complete genetic deletion of MCP-1 did not cause reduced macrophage numbers in the nerves due to compensatory proliferation of resident macrophages. In contrast, and as already seen in other CMT models, heterozygous deletion of MCP-1 led to reduced numbers of phagocytosing macrophages and an alleviation of demyelination. Whereas alleviated demyelination was transient, axonal damage was persistently improved and even robust axonal sprouting was detectable at 12 months. Other axon-related features were alleviated electrophysiological parameters, reduced muscle denervation and atrophy, and increased muscle strength. Similar to models for CMT1A and CMT1B, we identified MEK-ERK signalling as mediating MCP-1 expression in Cx32-deficient Schwann cells. Blocking this pathway by the inhibitor CI-1040 caused reduced MCP-1 expression, attenuation of macrophage increase and amelioration of myelin- and axon-related alterations. Thus, attenuation of MCP-1 upregulation by inhibiting ERK phosphorylation might be a promising approach to treat CMT1X and other so far untreatable inherited peripheral neuropathies in humans.

Mitosis of resident macrophages in the adult rat testis.

Resident macrophages are maintained at a comparatively high, yet stable, tissue concentration in the adult rat testis. After destruction of Leydig cells by ethane dimethane sulphonate treatment, the number of resident macrophages increases briefly and then decreases to below normal values, but returns to normal after the reappearance of Leydig cells. The mechanisms by which the adult testicular macrophage population is maintained, either by monocyte recruitment or by mitosis of the resident macrophages, have not been examined. An immunohistochemical dual labelling approach using a specific monoclonal antibody for resident macrophages, ED2, and markers of mitotic activity (bromodeoxyuridine incorporation and expression of the proliferating cell nuclear antigen) was used to investigate resident macrophage proliferation in Bouin's-fixed paraffin wax-embedded adult rat testes. Detection of the normally fixation sensitive antigen recognized by ED2 was achieved by using a decreased fixation time and antigen retrieval. Peaks of resident macrophage mitotic activity were observed during the phases of macrophage proliferation immediately after ethane dimethane sulphonate treatment and during the recovery phase associated with Leydig cell restoration. These data demonstrate that resident macrophages have the capacity to proliferate within the adult rat testis and, thus, this population of resident macrophages is maintained, at least in part, by mitotic division in situ.

In vivo responses of macrophages and myofibroblasts in the healing following isoproterenol-induced myocardial injury in rats.

To clarify the relation between macrophage and myofibroblast involvement in various myocardial diseases, the authors investigated the kinetics of these cells in the healing (scar tissue formation) following isoproterenol-induced myocardial injury in rats. Alpha-smooth muscle actin (alpha-SMA) expressing myofibroblasts were seen at the border of the affected area and appeared in the greatest numbers on days 3-7 post-injection, followed by a gradual decrease by day 35. The peak on day 3 was consistent with the timing of the highest proliferative activity of myofibroblasts. The number of ED1-positive macrophages began to increase as early as day 1, reaching a peak on day 3 within the injured myocardium. The expansion of ED1-positive macrophages preceded an increased number of alpha-SMA-positive myofibroblasts suggesting that myofibroblast proliferation and activation may be mediated by factors released by ED1-positive macrophages in response to myocardial injury. The number of ED2-positive tissue-fixed, resident macrophages gradually, increased from day 3 post-injection, and peaked on day 14, but the number of ED2-positive macrophages was consistently fewer than that of ED1-positive macrophages during the 35 day-observation period after the injection. The labelling index of the ED2-positive cells was maximal on day 14, indicative of local proliferation of resident macrophages. In the healing process after myocardial injury, ED1-positive macrophages increase markedly in the early stages: ED2-positive macrophages appear later.