Nitric oxide synthase 2 is required for conversion of pro-fibrogenic inflammatory CD133+ progenitors into F4/80+ macrophages in experimental autoimmune myocarditis.

AIMS: Experimental autoimmune myocarditis (EAM) model mirrors important mechanisms of inflammatory dilated cardiomyopathy (iDCM). In EAM, inflammatory CD133(+) progenitors are a major cellular source of cardiac myofibroblasts in the post-inflammatory myocardium. We hypothesized that exogenous delivery of macrophage-colony-stimulating factor (M-CSF) can stimulate macrophage lineage differentiation of inflammatory progenitors and, therefore, prevent their naturally occurring myofibroblast fate in EAM. METHODS AND RESULTS: EAM was induced in wild-type (BALB/c) and nitric oxide synthase 2-deficient (Nos2(-/-)) mice and CD133(+) progenitors were isolated from inflamed hearts. In vitro, M-CSF converted inflammatory CD133(+) progenitors into nitric oxide-producing F4/80(+) macrophages and prevented transforming growth factor-β-mediated myofibroblast differentiation. Importantly, only a subset of heart-infiltrating CD133(+) progenitors expresses macrophage-specific antigen F4/80 in EAM. These CD133(+)/F4/80(hi) cells show impaired myofibrogenic potential compared with CD133(+)/F4/80(-) cells. M-CSF treatment of wild-type mice with EAM at the peak of disease markedly increased CD133(+)/F4/80(hi) cells in the myocardium, and CD133(+) progenitors isolated from M-CSF-treated mice failed to differentiate into myofibroblasts. In contrast, M-CSF was not effective in converting CD133(+) progenitors from inflamed hearts of Nos2(-/-) mice into macrophages, and M-CSF treatment did not result in increased CD133(+)/F4/80(hi) cell population in hearts of Nos2(-/-) mice. Accordingly, M-CSF prevented post-inflammatory fibrosis and left ventricular dysfunction in wild-type but not in Nos2(-/-) mice. CONCLUSION: Active and NOS2-dependent induction of macrophage lineage differentiation abrogates the myofibrogenic potential of heart-infiltrating CD133(+) progenitors. Modulating the in vivo differentiation fate of specific progenitors might become a novel approach for the treatment of inflammatory heart diseases.

Effect of the host specific treatment in the phagocytosis of Trypanosoma cruzi blood forms by mouse peritoneal macrophages

Single doses of drugs active aginst Trypanosoma cruzi (megazol, nifurtimox and benznidazole) induce a rapid clearence of the blood parasites in experimentally infected mice. Furthermore, the in vitro phagocytosis and intracellular destruction by mouse peritoneal macrophage of blood forms collected from the treatment animals is strongly enhanced as compared with parasites from untreated controls. The uptake of the blood forms by macrophages is significantly higher with megazol than with benznidazole and nifurtimox, a finding that concurs with data showing that megazol is also the most active compound in the living host. The possibility that macrophages participate in a synergic effect between the host immune response and chemotherapeutic effect is discussed.

Macrophages from Crohn's disease patients exhibit deficient pro-repair functions

BACKGROUND: We previously reported that myeloid cells can induce mucosal healing in a mouse model of acute colitis. Promotion of mucosal repair is becoming a major goal in the treatment of Crohn's disease. Our aim in this study is to investigate the pro-repair function of myeloid cells in healthy donor (HD) and Crohn's disease patients (CD). METHODS: Peripheral blood mononuclear cells (PBMC) from HD and CD patients were isolated from blood samples by Ficoll density gradient. Monocytic CD14+ cells were positively selected by Macs procedure and then differentiated ex-vivo into macrophages (Mφ). The repair function of PBMC, CD14+ monocytic cells and macrophages were evaluated in an in vitro wound healing assay. RESULTS: PBMC and CD14+ myeloid cells from HD and CD were not able to repair at any tested cell concentration. Remarkably, HD Mφ were able to induce wound healing only at high concentration (105 added Mφ), but, if activated with heat killed bacteria, they were able to repair even at very low concentration. On the contrary, not activated CD Mφ were not able to promote healing at any rate, but this function was restored upon activation. CONCLUSION: We showed that CD Mφ in their steady state, unlike HD Mφ, are defective in promoting wound healing. Our results are in keeping with the current theory of CD as an innate immunodeficiency. Defective Mφ may be responsible to the mucosal repair defects in CD patients and to the subsequent chronic activation of the adaptive immune response.

NALP3 is not necessary for early protection against experimental tuberculosis.

In vitro, Toll-like receptors (TLR)2, 4 and 9 as well as NOD-like receptor 2 critically determine macrophage responses to Mycobacterium tuberculosis (Mtb) infection. However, in low-dose experimental murine tuberculosis, single or multiple deficiencies in TLRs 2, 4, 9 or NOD2 have little, if any, impact on early mycobacterial growth containment, granuloma formation and survival. Here, we analyzed the relevance of NALP3, one component of the danger-signaling inflammasome, for (i) Mtb-induced cytokine secretion in vitro and in vivo, (ii) restriction of Mtb replication in infected organs and (iii) granuloma formation. In the absence of functional NALP3, there was no IL-1beta and IL-18 production in Mtb-infected dendritic cells and macrophages in vitro, whereas secretion of IL-1alpha, IL-12p40 and TNF remained unaffected. After three weeks of infection, NALP3-deficient as well as IL-18-deficient mice were as capable as wildtype mice of restricting Mtb loads at a plateau level within well-differentiated granulomas. In conclusion, despite its involvement in cytokine processing, NALP3 is not essential for induction of protective immunity to Mtb.

Allergen-induced IgE-dependent gut inflammation in a human PBMC-engrafted murine model of allergy.

BACKGROUND: Humanized murine models comprise a new tool to analyze novel therapeutic strategies for allergic diseases of the intestine.¦OBJECTIVE: In this study we developed a human PBMC-engrafted murine model of allergen-driven gut inflammation and analyzed the underlying immunologic mechanisms.¦METHODS: Nonobese diabetic (NOD)-scid-γc(-/-) mice were injected intraperitoneally with human PBMCs from allergic donors together with the respective allergen or not. Three weeks later, mice were challenged with the allergen orally or rectally, and gut inflammation was monitored with a high-resolution video miniendoscopic system, as well as histologically.¦RESULTS: Using the aeroallergens birch or grass pollen as model allergens and, for some donors, also hazelnut allergen, we show that allergen-specific human IgE in murine sera and allergen-specific proliferation and cytokine production of human CD4(+) T cells recovered from spleens after 3 weeks could only be measured in mice treated with PBMCs plus allergen. Importantly, these mice had the highest endoscopic scores evaluating translucent structure, granularity, fibrin, vascularity, and stool after oral or rectal allergen challenge and a strong histologic inflammation of the colon. Analyzing the underlying mechanisms, we demonstrate that allergen-associated colitis was dependent on IgE, human IgE receptor-expressing effector cells, and the mediators histamine and platelet-activating factor.¦CONCLUSION: These results demonstrate that allergic gut inflammation can be induced in human PBMC-engrafted mice, allowing the investigation of pathophysiologic mechanisms of allergic diseases of the intestine and evaluation of therapeutic interventions.

An optimized method for establishing high purity murine CD8+ T cell cultures.

Establishing CD8(+) T cell cultures has been empirical and the published methods have been largely individual laboratory based. In this study, we optimized culturing conditions and show that IL-2 concentration is the most critical factor for the success of establishing CD8(+) T cell cultures. High IL-2 concentration encouraged T cells to non-specifically proliferate, express a B cell marker, B220, and undergo apoptosis. These cells also lose typical irregular T cell morphology and are incapable of sustaining long-term cultures. Using tetramer and intracellular cytokine assessments, we further demonstrated that many antigen-specific T cells have been rendered nonfunctional when expanded under high IL-2 concentration. When IL-2 is used in the correct range, B220-mediated cell depletion greatly enhanced the success rate of such T cell cultures.

Vaccination in murine schistosomiasis with adult worm derived antigens - II. Protective and immune response in inbred mice

Previous work in our laboratory, mainly foccused the prospects of achieving resistance against Schistosoma mansoni infection with adult worm-derived antigens in the form of a soluble extract (SE). This extract obtained by incubation of living adult schistosomes in saline, contains a large number of distinct molecules and was actually shown to be a significantly protective in different outbred animals models such as Swiss mice and rabbits. It thus appeared worthwile to investigate the potencial protective activity of SE in different inbred strains of mice, known to be highly susceptible to the infection. Herein we present data showing that DBA/2 mice, once immunized with SE acquire significant levels of resistance to a S. mansoni cercarial challenge. In addition, preliminary studies on the immune system of immunized animals reveled that, injection of SE caused no general inbalance of B or T cell responses.

Macrophages induce neutrophil apoptosis through membrane TNF, a process amplified by Leishmania major.

Neutrophils are recruited to the site of parasite inoculation within a few hours of infection with the protozoan parasite Leishmania major. In C57BL/6 mice, which are resistant to infection, neutrophils are cleared from the site of s.c. infection within 3 days, whereas they persist for at least 10 days in susceptible BALB/c mice. In the present study, we investigated the role of macrophages (MPhi) in regulating neutrophil number. Inflammatory cells were recruited by i.p. injection of either 2% starch or L. major promastigotes. Neutrophils were isolated and cultured in the presence of increasing numbers of MPhi. Extent of neutrophil apoptosis positively correlated with the number of MPhi added. This process was strictly dependent on TNF because MPhi from TNF-deficient mice failed to induce neutrophil apoptosis. Assays using MPhi derived from membrane TNF knock-in mice or cultures in Transwell chambers revealed that contact with MPhi was necessary to induce neutrophil apoptosis, a process requiring expression of membrane TNF. L. major was shown to exacerbate MPhi-induced apoptosis of neutrophils, but BALB/c MPhi were not as potent as C57BL/6 MPhi in this induction. Our results emphasize the importance of MPhi-induced neutrophil apoptosis, and membrane TNF in the early control of inflammation.

The role of egg antigens, cytokines in granuloma formation in murine schistosomiasis mansoni

The induction of granuloma formation by soluble egg antigens (SEA) of Schistosoma mansoni is accompanied by T cell-mediated lymphokine production that regulates the intensity of the response. In the present study we have examined the ability of SDS-PAGE fractioned SEA proteins to elicit granulomas and lymphokine production in infected and egg-immunized mice. At the acute stage of infection SEA fractions (<21, 25-30, 32-38, 60-66, 70-90, 93-125, and > 200 kD) that elicited pulmonary granulomas also elicited IL-2, IL-4 lymphokine production. At the chronic stage a diminished number of fractions (60-66, 70-90, 93-125, and > 200 kD) were able to elicit granulomas with an overall decrease in IL-2, IL-4 production. Granulomas were elicited by larval-egg crossreactive and egg-specific fractions at both the acute and chronic stage of the infection. Examination of lymphokine production from egg-immunized mice demonstrated that as early as 4 days IL-2 was produced by spleen cells stimulated with <21, 32-38, 40-46, 93-125, and >200 kD fractions. By 16 days, IL-2production was envoked by 8 of 9 fractions. IL-4 production at 4 days in response to all fractions was minimal while at 16 days IL-4 was elicited with the < 21, 25-30, 50-56, 93-125, and > 200 kD fractions. The present study reveals differences in the range of SEA fractions able to elicit granulomas and IL-2, IL-4 production between acute and chronic stages of infection. Additionally, this study demonstrates sequential (IL-2 followed by IL-4) lymphokine production during the primary egg antigen response.

Experimental murine schistosomiasis mansoni: estabilishment of the chronic phase of the disease

After the acute hyperergic phase of schistosomal infection, the chronic phase of the disease corresponds to the estabilishment of a relative equilibrium between the host and the parasite. This involves: (1) A shift from the predominance of the TH2 response observed in the acute phase, to the predominance of the TH1 response in the chronic phase of the disease, with modification of lymphokine and immunoglobulin secretions patterns. (2) Redistribution of hosts responses to parasite, with predominance of systemic controls in the acute phase, and a shift towards local tissue responses in the chronic phase. This redistribution relieves the hyperergic responses involving the whole body of the host, and delimits cellular and molecular reactions to parasites to only those tissues that are directly involved by the adult parasites and their eggs. Mobilization of eosinophil granulocytes in schistosomal periovular granulomas is one of examples of this redistribution.

Molecular detection and identification of zygomycetes species from paraffin-embedded tissues in a murine model of disseminated zygomycosis: a collaborative European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Fungal Infection Study Group (EFISG) evaluation.

The present study was performed to assess the interlaboratory reproducibility of the molecular detection and identification of species of Zygomycetes from formalin-fixed paraffin-embedded kidney and brain tissues obtained from experimentally infected mice. Animals were infected with one of five species (Rhizopus oryzae, Rhizopus microsporus, Lichtheimia corymbifera, Rhizomucor pusillus, and Mucor circinelloides). Samples with 1, 10, or 30 slide cuts of the tissues were prepared from each paraffin block, the sample identities were blinded for analysis, and the samples were mailed to each of seven laboratories for the assessment of sensitivity. A protocol describing the extraction method and the PCR amplification procedure was provided. The internal transcribed spacer 1 (ITS1) region was amplified by PCR with the fungal universal primers ITS1 and ITS2 and sequenced. As negative results were obtained for 93% of the tissue specimens infected by M. circinelloides, the data for this species were excluded from the analysis. Positive PCR results were obtained for 93% (52/56), 89% (50/56), and 27% (15/56) of the samples with 30, 10, and 1 slide cuts, respectively. There were minor differences, depending on the organ tissue, fungal species, and laboratory. Correct species identification was possible for 100% (30 cuts), 98% (10 cuts), and 93% (1 cut) of the cases. With the protocol used in the present study, the interlaboratory reproducibility of ITS sequencing for the identification of major Zygomycetes species from formalin-fixed paraffin-embedded tissues can reach 100%, when enough material is available.