Fibroblast stimulation in schistosomiasis. IV. Isolated egg granulomas elaborate a fibroblast chemoattractant in vitro

Hepatic fibrosis complicates the chronic granulomatous inflammatory reaction to Schistosoma mansoni eggs, and is the major cause of morbidity and mortality in human schistosomiasis. We previously presented evidence that schistosomal egg granulomas secreted factors that can stimulate fibroblast proliferation and collagen synthesis in vitro. We now report that serum-free supernatants from cultures of hepatic egg granulomas isolated from S. mansoni-infected mice contained activity that stimulated the directional migration of human and guinea pig dermal fibroblasts in modified Boyden chambers. This fibroblast chemotactic activity was also detected in culture supernatants of granuloma adherent cells highly enriched for macrophages (95% latex-ingesting) but not in culture supernatants from resident peritoneal macrophages of uninfected or infected mice. This suggests that granuloma macrophages are a source of the chemotactic activity. The chemoattractant had the properties of large molecular weight (greater than 200,000 daltons; Sephadex G-200 gel filtration), pl approximately 4.5 (preparative flatbed isoelectrofocusing in granular matrix), heat stability (56 degrees C; 45 min), and trypsin sensitivity. Since preincubation of the partially purified granuloma and adherent-cell derived chemoattractants with rabbit anti-human fibronectin antibody abolished their chemotactic activity, it appears that the factor is antigenically similar to fibronectin. We propose that egg granuloma macrophages are activated in vivo to secrete a fibronectin-like molecule with activity that stimulates the directional migration of fibroblasts. This factor may therefore play a role in the local recruitment of fibroblasts and, in concert with other granuloma-derived factors, may play an important role in the pathogenesis of hepatic fibrosis in schistosomiasis.     

Variability in Tuberculosis Granuloma T Cell Responses Exists,but a Balance of Pro- and Anti-inflammatory Cytokines Is Associated with Sterilization

Lung granulomas are the pathologic hallmark of tuberculosis (TB). T cells are a major cellular component of TB lung granulomas and are known to play an important role in containment of Mycobacterium tuberculosis (Mtb) infection. We used cynomolgus macaques, a non-human primate model that recapitulates human TB with clinically active disease, latent infection or early infection, to understand functional characteristics and dynamics of T cells in individual granulomas. We sought to correlate T cell cytokine response and bacterial burden of each granuloma, as well as granuloma and systemic responses in individual animals. Our results support that each granuloma within an individual host is independent with respect to total cell numbers, proportion of T cells, pattern of cytokine response, and bacterial burden. The spectrum of these components overlaps greatly amongst animals with different clinical status, indicating that a diversity of granulomas exists within an individual host. On average only about 8% of T cells from granulomas respond with cytokine production after stimulation with Mtb specific antigens, and few “multi-functional” T cells were observed. However, granulomas were found to be “multi-functional” with respect to the combinations of functional T cells that were identified among lesions from individual animals. Although the responses generally overlapped, sterile granulomas had modestly higher frequencies of T cells making IL-17, TNF and any of T-1 (IFN-γ, IL-2, or TNF) and/or T-17 (IL-17) cytokines than non-sterile granulomas. An inverse correlation was observed between bacterial burden with TNF and T-1/T-17 responses in individual granulomas, and a combinatorial analysis of pair-wise cytokine responses indicated that granulomas with T cells producing both pro- and anti-inflammatory cytokines (e.g. IL-10 and IL-17) were associated with clearance of Mtb. Preliminary evaluation suggests that systemic responses in the blood do not accurately reflect local T cell responses within granulomas.     

Hepatic granulomas induced by Schistosoma mansoni in mice deficient for connexin 43 present lower cell proliferation and higher collagen content

Granuloma formation involves a coordinated interaction between monocytes and macrophages, epithelioid cells, lymphocytes, eosinophils, neutrophils and fibroblasts. It has been established that extracellular communication via cytokines is important for the assembly of granulomas. However, the importance of gap junctions and intercellular communication to granuloma formation and development had never been assessed. Connexins are proteins that form gap junctions, and connexin 43 (Cx43) is present in macrophages, lymphoid cells, myelogenous cells, fibroblasts and others. We analyzed the effect of heterologous deletion of Gja1 (Cx43 gene) on the formation and development of hepatic granulomas induced by Schistosoma mansoni eggs. Heterozygous (Cx43(+/-)) and wild-type (Cx43(+/+)) mice were infected subcutaneously with S. mansoni cercarie and evaluated after 6, 8 and 12 weeks. Granuloma cells express Cx43, as revealed by real-time PCR in isolated granulomas, and by immunohistochemistry. Cx43 expression was reduced in Cx43(+/-) mice, as expected. No differences in the average area of granulomas or number of cells per granuloma were observed between mice of different genotypes. However, granuloma cells from Cx43(+/-) mice displayed a reduced index of the proliferating cell nuclear antigen (PCNA) labeling at 8 and 12 weeks post-infection. Moreover, Cx43(+/-) granulomas unexpectedly presented a higher degree of fibrosis, quantified by morphometric analysis in Sirius Red-stained slides. Our results indicate that the deletion of one allele of the Cx43 gene, and possibly the reduced gap junction intercellular communication capacity (GJIC), may impair the interactions between granuloma cells, reducing their proliferation and increasing their collagen content, thereby modifying the characteristics of S. mansoni granuloma in mice.     

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.     

Fibroblast stimulation in schistosomiasis. V. Egg granuloma macrophages spontaneously secrete a fibroblast-stimulating factor

Isolated intact egg granulomas from the liver of Schistosoma mansoni-infected mice have been previously shown to elaborate factors in vitro that can stimulate fibroblasts for biological functions that are of potential importance in the pathogenesis of hepatic fibrosis in schistosomiasis. We report here that cell cultures obtained from monodispersed granuloma cell suspensions, and specifically enriched for macrophages (95% to 100%) spontaneously elaborated fibroblast proliferation-stimulating activity in vitro. These cells possessed functional and phenotyptic characteristics of activated macrophages. In contrast, control peritoneal macrophages from uninfected mice lacked such phenotypic characteristics, and did not spontaneously elaborate fibrogenic activity in vitro. The granuloma macrophage activity was present, pre-formed within the isolated cells, and was continuously elaborated during 72 hr of incubation. By gel infiltration chromatography (Sephacryl S-200 sf), fibroblast-stimulating activity was identified in two pooled fractions, one with estimated molecular radius (Mr) of 46 kd to 57 kd and the other with Mr of 10 kd to 16 kd. Preparative isoelectric focusing in granular gel of crude macrophage culture supernatants identified peak activity in fractions with pI approximately 5. Two different serine esterase inhibitors had no effect on the ability of crude granuloma macrophage supernatants to stimulate fibroblast proliferation. Whereas crude and chromatographed fractions of granuloma macrophage supernatant were active for fibroblasts, they had minimal or no interleukin 1 (IL 1) activity when tested in a thymocyte proliferation assay. In contrast, resident peritoneal macrophages from the same infected mice spontaneously secreted substantial IL 1 and fibroblast-stimulating activity in vitro. We conclude that egg granuloma macrophages are activated in vivo to secrete fibrogenic molecules functionally distinct from IL 1, which might contribute to the pathogenesis of hepatic fibrosis in schistosomiasis.     

Temporal Expression of Chemokines Dictates the Hepatic Inflammatory Infiltrate in a Murine Model of Schistosomiasis

Schistosomiasis continues to be an important cause of parasitic morbidity and mortality world-wide. Determining the molecular mechanisms regulating the development of granulomas and fibrosis will be essential for understanding how schistosome antigens interact with the host environment. We report here the first whole genome microarray analysis of the murine liver during the progression of Schistosoma japonicum egg-induced granuloma formation and hepatic fibrosis. Our results reveal a distinct temporal relationship between the expression of chemokine subsets and the recruitment of cells to the infected liver. Genes up-regulated earlier in the response included T- and B-cell chemoattractants, reflecting the early recruitment of these cells illustrated by flow cytometry. The later phases of the response corresponded with peak recruitment of eosinophils, neutrophils, macrophages and myofibroblasts/hepatic stellate cells (HSCs) and the expression of chemokines with activity for these cells including CCL11 (eotaxin 1), members of the Monocyte-chemoattractant protein family (CCL7, CCL8, CCL12) and the Hepatic Stellate Cell/Fibrocyte chemoattractant CXCL1. Peak expression of macrophage chemoattractants (CCL6, CXCL14) and markers of alternatively activated macrophages (e.g. Retnla) during this later phase provides further evidence of a role for these cells in schistosome-induced pathology. Additionally, we demonstrate that CCL7 immunolocalises to the fibrotic zone of granulomas. Furthermore, striking up-regulation of neutrophil markers and the localisation of neutrophils and the neutrophil chemokine S100A8 to fibrotic areas suggest the involvement of neutrophils in S. japonicum-induced hepatic fibrosis. These results further our understanding of the immunopathogenic and, especially, chemokine signalling pathways that regulate the development of S. japonicum-induced granulomas and fibrosis and may provide correlative insight into the pathogenesis of other chronic inflammatory diseases of the liver where fibrosis is a common feature.     

Tuberculous granuloma formation is enhanced by a mycobacterium virulence determinant

Granulomas are organized host immune structures composed of tightly interposed macrophages and other cells that form in response to a variety of persistent stimuli, both infectious and noninfectious. The tuberculous granuloma is essential for host containment of mycobacterial infection, although it does not always eradicate it. Therefore, it is considered a host-beneficial, if incompletely efficacious, immune response. The Mycobacterium RD1 locus encodes a specialized secretion system that promotes mycobacterial virulence by an unknown mechanism. Using transparent zebrafish embryos to monitor the infection process in real time, we found that RD1-deficient bacteria fail to elicit efficient granuloma formation despite their ability to grow inside of infected macrophages. We showed that macrophages infected with virulent mycobacteria produce an RD1-dependent signal that directs macrophages to aggregate into granulomas. This Mycobacterium-induced macrophage aggregation in turn is tightly linked to intercellular bacterial dissemination and increased bacterial numbers. Thus, mycobacteria co-opt host granulomas for their virulence.     

GSDMD-dependent neutrophil extracellular traps promote macrophage-to-myofibroblast transition and renal fibrosis in obstructive nephropathy

Renal fibrosis is a common consequence of various progressive nephropathies, including obstructive nephropathy, and ultimately leads to kidney failure. Infiltration of inflammatory cells is a prominent feature of renal injury after draining blockages from the kidney, and correlates closely with the development of renal fibrosis. However, the underlying molecular mechanism behind the promotion of renal fibrosis by inflammatory cells remains unclear. Herein, we showed that unilateral ureteral obstruction (UUO) induced Gasdermin D (GSDMD) activation in neutrophils, abundant neutrophil extracellular traps (NETs) formation and macrophage-to-myofibroblast transition (MMT) characterized by α-smooth muscle actin (α-SMA) expression in macrophages. Gsdmd deletion significantly reduced infiltration of inflammatory cells in the kidneys and inhibited NETs formation, MMT and thereby renal fibrosis. Chimera studies confirmed that Gsdmd deletion in bone marrow-derived cells, instead of renal parenchymal cells, provided protection against renal fibrosis. Further, specific deletion of Gsdmd in neutrophils instead of macrophages protected the kidney from undergoing fibrosis after UUO. Single-cell RNA sequencing identified robust crosstalk between neutrophils and macrophages. In vitro, GSDMD-dependent NETs triggered p65 translocation to the nucleus, which boosted the production of inflammatory cytokines and α-SMA expression in macrophages by activating TGF-β1/Smad pathway. In addition, we demonstrated that caspase-11, that could cleave GSDMD, was required for NETs formation and renal fibrosis after UUO. Collectively, our findings demonstrate that caspase-11/GSDMD-dependent NETs promote renal fibrosis by facilitating inflammation and MMT, therefore highlighting the role and mechanisms of NETs in renal fibrosis.Subject terms: Obstructive nephropathy, Inflammation     

Foamy Macrophages from Tuberculous Patients' Granulomas Constitute a Nutrient-Rich Reservoir for M. tuberculosis Persistence

Tuberculosis (TB) is characterized by a tight interplay between Mycobacterium tuberculosis and host cells within granulomas. These cellular aggregates restrict bacterial spreading, but do not kill all the bacilli, which can persist for years. In-depth investigation of M. tuberculosis interactions with granuloma-specific cell populations are needed to gain insight into mycobacterial persistence, and to better understand the physiopathology of the disease. We have analyzed the formation of foamy macrophages (FMs), a granuloma-specific cell population characterized by its high lipid content, and studied their interaction with the tubercle bacillus. Within our in vitro human granuloma model, M. tuberculosis long chain fatty acids, namely oxygenated mycolic acids (MA), triggered the differentiation of human monocyte-derived macrophages into FMs. In these cells, mycobacteria no longer replicated and switched to a dormant non-replicative state. Electron microscopy observation of M. tuberculosis–infected FMs showed that the mycobacteria-containing phagosomes migrate towards host cell lipid bodies (LB), a process which culminates with the engulfment of the bacillus into the lipid droplets and with the accumulation of lipids within the microbe. Altogether, our results suggest that oxygenated mycolic acids from M. tuberculosis play a crucial role in the differentiation of macrophages into FMs. These cells might constitute a reservoir used by the tubercle bacillus for long-term persistence within its human host, and could provide a relevant model for the screening of new antimicrobials against non-replicating persistent mycobacteria.     

Identifying control mechanisms of granuloma formation during M. tuberculosis infection using an agent-based model

Infection with Mycobacterium tuberculosis is a major world health problem. An estimated 2 billion people are presently infected and the disease causes approximately 3 million deaths per year. After bacteria are inhaled into the lung, a complex immune response is triggered leading to the formation of multicellular structures termed granulomas. It is believed that the collection of host granulomas either contain bacteria resulting in a latent infection or are unable to do so, leading to active disease. Thus, understanding granuloma formation and function is essential for improving both diagnosis and treatment of tuberculosis. Granuloma formation is a complex spatio-temporal system involving interactions of bacteria, specific immune cells, including macrophages, CD4+ and CD8+ T cells, as well as immune effectors such as chemokine and cytokines. To study this complex dynamical system we have developed an agent-based model of granuloma formation in the lung. This model combines continuous representations of chemokines with discrete agent representations of macrophages and T cells in a cellular automata-like environment. Our results indicate that key host elements involved in granuloma formation are chemokine diffusion, prevention of macrophage overcrowding within the granuloma, arrival time, location and number of T cells within the granuloma, and an overall host ability to activate macrophages. Interestingly, a key bacterial factor is its intracellular growth rate, whereby slow growth actually facilitates survival.     

Mycobacterium tuberculosis-Induced Polarization of Human Macrophage Orchestrates the Formation and Development of Tuberculous Granulomas In Vitro

The tuberculous granuloma is an elaborately organized structure and one of the main histological hallmarks of tuberculosis. Macrophages, which are important immunologic effector and antigen-presenting cells, are the main cell type found in the tuberculous granuloma and have high plasticity. Macrophage polarization during bacterial infection has been elucidated in numerous recent studies; however, macrophage polarization during tuberculous granuloma formation and development has rarely been reported. It remains to be clarified whether differences in the activation status of macrophages affect granuloma formation. In this study, the variation in macrophage polarization during the formation and development of tuberculous granulomas was investigated in both sections of lung tissues from tuberculosis patients and an in vitro tuberculous granuloma model. The roles of macrophage polarization in this process were also investigated. Mycobacterium tuberculosis (M. tuberculosis) infection was found to induce monocyte-derived macrophage polarization. In the in vitro tuberculous granuloma model, macrophage transformation from M1 to M2 was observed over time following M. tuberculosis infection. M2 macrophages were found to predominate in both necrotic and non-necrotic granulomas from tuberculosis patients, while both M1 and M2 polarized macrophages were found in the non-granulomatous lung tissues. Furthermore, it was found that M1 macrophages promote granuloma formation and macrophage bactericidal activity in vitro, while M2 macrophages inhibit these effects. The findings of this study provide insights into the mechanism by which M. tuberculosis circumvents the host immune system as well as a theoretical foundation for the development of novel tuberculosis therapies based on reprogramming macrophage polarization.     

cGAS exacerbates Schistosoma japonicum infection in a STING-type I IFN-dependent and independent manner

Schistosomiasis, which is caused by infection with Schistosoma spp., is characterized by granuloma and fibrosis in response to egg deposition. Pattern recognition receptors are important to sense invading Schistosoma, triggering an innate immune response, and subsequently shaping adaptive immunity. Cyclic GMP-AMP synthase (cGAS) was identified as a major cytosolic DNA sensor, which catalyzes the formation of cyclic GMP-AMP (cGAMP), a critical second messenger for the activation of the adaptor protein stimulator of interferon genes (STING). The engagement of STING by cGAMP leads to the activation of TANK-binding kinase 1 (TBK1), interferon regulatory factor 3 (IRF3), and the subsequent type I interferon (IFN) response. cGAS is suggested to regulate infectious diseases, autoimmune diseases, and cancer. However, the function of cGAS in helminth infection is unclear. In this study, we found that Cgas deficiency enhanced the survival of mice infected with S. japonicum markedly, without affecting the egg load in the liver. Consistently, Cgas deletion alleviated liver pathological impairment, reduced egg granuloma formation, and decreased fibrosis severity. In contrast, Sting deletion reduced the formation of egg granulomas markedly, but not liver fibrosis. Notably, Cgas or Sting deficiency reduced the production of IFNβ drastically in mice infected with S. japonicum. Intriguingly, intravenous administration of recombinant IFNβ exacerbated liver damage and promoted egg granuloma formation, without affecting liver fibrosis. Clodronate liposome-mediated depletion of macrophages indicated that macrophages are the major type of cells contributing to the induction of the type I IFN response during schistosome infection. Moreover, cGAS is important for type I IFN production and phosphorylation of TBK1 and IRF3 in response to stimulation with S. japonicum egg- or adult worm-derived DNA in macrophages. Our results clarified the immunomodulatory effect of cGAS in the regulation of liver granuloma formation during S. japonicum infection, involving sensing schistosome-derived DNA and producing type I IFN. Additionally, we showed that cGAS regulates liver fibrosis in a STING-type I–IFN-independent manner.     

An in vitro model of the leukocyte interactions associated with granuloma formation in Mycobacterium tuberculosis infection

The principal defense of the human host against a Mycobacterium tuberculosis infection is the formation of granulomas, organized collections of activated macrophages, including epithelioid and multinucleated giant cells, surrounded by lymphocytes. This granuloma can sequester and contain the bacteria preventing active disease, and if the granuloma is maintained, these bacteria may remain latent for a person's lifetime. Secretion of a variety of chemoattractant cytokines following phagocytosis of the bacilli by the macrophage is critical not only to the formation of the granuloma but also to its maintenance. To investigate this process of early granuloma formation, we developed an in vitro model composed entirely of human cells. Combining blood lymphocytes and autologous macrophages from healthy purified protein derivative skin test-negative individuals and mycobacteria resulted in the formation of small, rounded aggregate structures. Microscopic examination found macrophage-specific CD68(+) epithelioid macrophages and small round CD3(+) lymphocytes that in complex resembled small granulomas seen in clinical pathology specimens. Acid-fast staining bacteria were observed between and possibly within the cells composing the granulomas. Supernatants from the infected cells collected at 24 and 48 h and 5 and 9 days after infection were analyzed by a multiplexed cytokine bead-based assay using the Luminex 100 and were found to contain interleukin (IL)-6, IL-8, interferon-gamma and tumor necrosis factor-alpha, cytokines known to be involved in human granuloma formation, in quantities from two-fold to 7000-fold higher than supernatants from uninfected control cells. In addition, chemotaxis assays demonstrated that the same supernatants attracted significantly more human peripheral blood mononuclear cells than those of uninfected cells (P<0.001). This model may provide insight into the earliest stages of granuloma formation in those newly infected.     

Differentiation,Distribution and γδ T Cell-Driven Regulation of IL-22-Producing T Cells in Tuberculosis

Differentiation, distribution and immune regulation of human IL-22-producing T cells in infections remain unknown. Here, we demonstrated in a nonhuman primate model that M. tuberculosis infection resulted in apparent increases in numbers of T cells capable of producing IL-22 de novo without in vitro Ag stimulation, and drove distribution of these cells more dramatically in lungs than in blood and lymphoid tissues. Consistently, IL-22-producing T cells were visualized in situ in lung tuberculosis (TB) granulomas by confocal microscopy and immunohistochemistry, indicating that mature IL-22-producing T cells were present in TB granuloma. Surprisingly, phosphoantigen HMBPP activation of Vγ2Vδ2 T cells down-regulated the capability of T cells to produce IL-22 de novo in lymphocytes from blood, lung/BAL fluid, spleen and lymph node. Up-regulation of IFNγ-producing Vγ2Vδ2 T effector cells after HMBPP stimulation coincided with the down-regulated capacity of these T cells to produce IL-22 de novo. Importantly, anti-IFNγ neutralizing Ab treatment reversed the HMBPP-mediated down-regulation effect on IL-22-producing T cells, suggesting that Vγ2Vδ2 T-cell-driven IFNγ-networking function was the mechanism underlying the HMBPP-mediated down-regulation of the capability of T cells to produce IL-22. These novel findings raise the possibility to ultimately investigate the function of IL-22 producing T cells and to target Vγ2Vδ2 T cells for balancing potentially hyper-activating IL-22-producing T cells in severe TB.     

Schistosome eggs stimulate reactive oxygen species production to enhance M2 macrophage differentiation and promote hepatic pathology in schistosomiasis

Schistosomiasis is a neglected tropical disease of public health concern. The most devastating pathology in schistosomiasis japonica and mansoni is mainly attributed to the egg-induced granulomatous response and secondary fibrosis in host liver, which may lead to portal hypertension or even death of the host. Schistosome eggs induce M2 macrophages-rich granulomas and these M2 macrophages play critical roles in the maintenance of granuloma and subsequent fibrosis. Reactive oxygen species (ROS), which are highly produced by stimulated macrophages during infection and necessary for the differentiation of M2 macrophages, are massively distributed around deposited eggs in the liver. However, whether ROS are induced by schistosome eggs to subsequently promote M2 macrophage differentiation, and the possible underlying mechanisms as well, remain to be clarified during S. japonicum infection. Herein, we observed that extensive expression of ROS in the liver of S. japonicum-infected mice. Injection of ROS inhibitor in infected mice resulted in reduced hepatic granulomatous responses and fibrosis. Further investigations revealed that inhibition of ROS production in S. japonicum-infected mice reduces the differentiation of M2, accompanied by increased M1 macrophage differentiation. Finally, we proved that S. japonicum egg antigens (SEA) induce a high level of ROS production via both nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) and mitochondria in macrophages. Our study may help to better understand the mechanism of schistosomiasis japonica-induced hepatic pathology and contribute to the development of potential therapeutic strategies by interfering with ROS production.     

A Petri Net Model of Granulomatous Inflammation: Implications for IL-10 Mediated Control of Leishmania donovani Infection

Experimental visceral leishmaniasis, caused by infection of mice with the protozoan parasite Leishmania donovani, is characterized by focal accumulation of inflammatory cells in the liver, forming discrete “granulomas” within which the parasite is eventually eliminated. To shed new light on fundamental aspects of granuloma formation and function, we have developed an in silico Petri net model that simulates hepatic granuloma development throughout the course of infection. The model was extensively validated by comparison with data derived from experimental studies in mice, and the model robustness was assessed by a sensitivity analysis. The model recapitulated the progression of disease as seen during experimental infection and also faithfully predicted many of the changes in cellular composition seen within granulomas over time. By conducting in silico experiments, we have identified a previously unappreciated level of inter-granuloma diversity in terms of the development of anti-leishmanial activity. Furthermore, by simulating the impact of IL-10 gene deficiency in a variety of lymphocyte and myeloid cell populations, our data suggest a dominant local regulatory role for IL-10 produced by infected Kupffer cells at the core of the granuloma.     

Adherent-invasive Escherichia coli isolated from Crohn's disease patients induce granulomas in vitro

Adherent-invasive Escherichia coli (AIEC) have been shown to be highly associated with ileal Crohn's disease (CD). AIEC survive within infected macrophages, residing within the phagolysosomal compartment where they take advantage of the low pH to replicate extensively. We investigated whether, like the tuberculous bacillus which also persists within macrophages, AIEC LF82 induces the formation of granulomas, which are a common histopathological feature of CD. For this purpose, we have taken advantage of an in vitro model of human granulomas that we recently developed, based on blood-derived mononuclear cells. We demonstrated that AIEC LF82 induces aggregation of infected macrophages, fusion of some of them to form multinucleated giant cells and subsequent recruitment of lymphocytes. Light microscopy and scanning electron microscopy analysis of the cell aggregates confirmed their granuloma features. This was further confirmed by histological analysis of granuloma sections. Noteworthy, this phenomenon can be reproduced by soluble protein extracts of AIEC LF82 coated onto beads. Although the cell aggregates not completely mimic natural CD-associated granulomas, they are very similar to early stages of epithelioid granulomas.     

Release of soluble factors from lymph nodes containing mycobacterial granulomas and their effect on fibroblast function in vitro

A study has been made of the action of culture supernatants from guinea pig lymph nodes containing mycobacterial granulomas on protein and DNA synthesis of homologous fibroblast cultures. Supernatants from both the Bacillus Calmette-Guerin (BCG) and Mycobacterium leprae granulomas release soluble nondialysable factors in vitro which stimulate [¹⁴C]proline and [¹⁴C]leucine incorporation by fibroblasts and depress their [³H]thymidine uptake. These supernatants did not show any detectable migratory inhibitory activity in vitro. On the other hand, supernatants from sensitized lymphocytes incubated with purified protein derivative (positive migratory inhibitory activity) had no effect on fibroblast function. Thus, the effect of granuloma supernatants is unlikely to be due to lymphokines. However, supernatants from dinitrofluorobenzene-sensitized lymph nodes also showed a stimulation of [¹⁴C]proline incorporation into total protein synthesised by fibroblasts and depressed the [³H]thymidine uptake. Furthermore, supernatants from live BCG organisms in culture on addition to fibroblasts enhanced their [³H]thymidine uptake in vitro. It would appear therefore that fibroblast activation in lymph nodes containing mycobacterial granulomas could result from the release of soluble factors of lymphocyte origin rather than from cells of the mononuclear phagocyte system. These factors appear to be independent of classical lymphokines that act on macrophages in vitro. An additional factor may be derived from the mycobacteria themselves.