Amélia Sarmento

The role of macrophage activation and of Bcg-encoded macrophage function(s) in the control of Mycobacterium avium infection in mice.

Following the intraperitoneal inoculation of 2.5 × 108 colony‐forming units of Mycobacterium avium strain ATCC 25291, there was bacillary growth in the liver, spleen and peritoneal cavity of C57BL/6, C57BL/10, DBA/1 and BALB/c mice whereas DBA/2, C3H/He, CBA/Ca and CD‐1 mice controlled the infection showing constant or slightly decreasing numbers of viable bacteria in the liver and spleen and effective clearance of the bacilli from the peritoneal cavities. The acquisition of non‐specific resistance (NSR) to Listeria monocytogenes during the infection by M. avium was high in C57BL/6, BALB/c and C3H/He mice and negligible in DBA/2 and CD‐1 mice. The magnitude of the acquisition of NSR was reduced in T cell‐deficient mice and was directly proportional to the dose of the inoculum of M. avium. The production of hydrogen peroxide by phorbol myristate acetate‐stimulated peritoneal macrophages of M. avium‐infected mice was higher in C57BL/6 and BALB/c mice than in CD‐1, DBA/2 and C3H/He animals. BALB/c. Begr (C.D2) mice, unlike their congenic strain BALB/c, restricted bacterial growth following the intravenous inoculation of 2.5 × 108 CFU of M. avium as efficiently as DBA/2 mice. C.D2 and BALB/c peritoneal macrophages from infected mice produced similar amounts of H2O2 but BALB/c mice developed higher levels of NSR to listeria than C.D2 mice. The production of nitrite by peritoneal macrophages from infected mice was found to be enhanced in DBA/2 and C3H/He but not in BALB/c, C57BL/6, CD‐1 and C.D2 mice. Resident peritoneal macrophages from C.D2 mice were more bacteriostatic in vitro for M. avium than macrophages from BALB/c mice. The same relative differences between the two macrophage populations were observed when the cells were activated with lymphokines. The results show that the resistance to M. avium infection in mice is under the control of the Bcg gene and that susceptibility may be due to some defect in macrophage antibacterial function not completely overcome by the activation of this phagocyte in the susceptible strains of mice.

Tumour necrosis factor-alpha (TNF-α) in the host resistance to mycobacteria of distinct virulence

The relative virulence of different isolates of Mycobacterium avium has been linked to their capacity to trigger the secretion of TNF from the macrophages they infect. Smooth opaque (SmOp) variants of Myco. avium have been shown to trigger higher expression of TNF‐α by macrophages in vitro than the smooth transparent (SmTr) variants. To analyse the role of TNF in resistance to infection by Myco. avium, we studied the infection by two different morphotypes of strain 2.151 of Myco. avium both in vitro and in vivo in the presence or absence of neutralizing antibodies to TNF. No effects were found in vitro regarding the growth of either isolate of Myco. avium. In vivo, only the virulent SmTr morphotype showed enhanced growth in the presence of the neutralizing antibodies. This enhancement occurred relatively late when priming for TNF secretion in vivo was evident. Among four isolates of Myco. avium, three virulent ones induced a marked priming for TNF release and one avirulent strain did not. Mycobacterium tuberculosis H37Ra, which is very active in inducing TNF release due to its lipoarabinomannan moiety, was used to compare with the previous results. The growth of H37Ra in macrophages was increased in vitro by the neutralization of TNF and neutralization of either TNF and/or interferon‐gamma (IFN‐γ) enhanced the in vivo proliferation of this microbe in the spleen and liver of infected animals, whereas only the combination of both anti‐TNF and anti‐IFN‐γ enhanced bacterial proliferation in the lung. We conclude that resistance to the avirulent strains of Myco. avium did not involve TNF, but rather antimicrobial mechanisms expressed consitutively in the mononuclear phagocytes. In contrast, TNF plays an important role in the control of Myco. tuberculosis H37Ra infection.

Modulation of the activity of sea bass (Dicentrarchus labrax) head-kidney macrophages by macrophage activating factor(s) and lipopolysaccharide.

The aim of this study was to establish the requirements for macrophage activating factor (MAF) production by sea bass head-kidney leucocytes and the kinetics of macrophage activation when exposed to MAF-containing supernatants and/or lipopolysaccharide (LPS), a known macrophage stimulant. MAF activity was found in culture supernatants of total head-kidney leucocytes pulsed with 5 microg ml(-1)Con A, 5 or 10 ng ml(-1)PMA and 100 ng ml(-1)calcium ionophore, or 10 microg ml(-1)Con A alone, as assessed by the capacity to prime macrophages for enhanced production of reactive oxygen intermediates (ROI). Mixed leucocyte cultures from two or eight fish showed higher MAF activity after stimulation, indicating that a mixed leucocyte reaction was also important for MAF production. MAF-induced activation of macrophage cultures was highest at 18 h of exposure and was lost by 72 h except for MAF induced by Con A-stimulation alone. LPS primed macrophages for increased ROI production at early incubation times and down-regulated ROI production after 24 h. LPS had no effect in further stimulating the MAF-induced priming effect on production of ROI and down-regulated the MAF-priming by 48 h. Sea bass head-kidney macrophages did not show increased nitrite production when exposed to MAF and/or LPS, which may be related to their differentiation status.

Epstein-Barr virus in inflammatory bowel disease-correlation with different therapeutic regimens.

Background: Inflammatory bowel disease (IBD) is associated with a higher prevalence of opportunistic infections. Epstein–Barr virus (EBV) is a ubiquitous virus related to several malignancies, namely lymphoma; its prevalence in patients with IBD and its relation with different therapeutic regimens are not well studied. Methods: Patients followed in our IBD outpatient clinic were consecutively enrolled for participation in a prospective study, and healthy volunteers were recruited as controls. EBV DNA was measured at least 1 time in each patient. Results: Three hundred and seventy-nine individuals were enrolled in the study (93 treated with 5-aminosalicylates, 91 with azathioprine, 70 with infliximab, 43 with combined treatment with infliximab and azathioprine, and 82 controls). More than 90% of the patients had previous EBV exposure. EBV DNA was found in 132 samples (35%); its prevalence was significantly higher in every group of patients with IBD, comparing to controls. Among patients with IBD, infliximab with or without azathioprine was related to higher prevalence of EBV comparing to azathioprine alone or 5-aminosalicylates (P < 0.05). Age above 60 years was related to EBV DNA positivity with a specificity of 92%. Concerning treated groups, ulcerative colitis was the only risk factor identified for high levels of EBV DNA (>1000 and 2500 copies per milliliter). No relationship was found between EBV and C-reactive protein. Conclusions: IBD is a risk factor for the presence of EBV DNA in blood, particularly in older patients and in those taking infliximab. C-reactive protein was not related to EBV DNA prevalence.

Macrophages from IBD patients exhibit defective tumour necrosis factor-α secretion but otherwise normal or augmented pro-inflammatory responses to infection.

Defects in macrophage function have been implicated in the establishment of Crohns disease (CD). However, the response of macrophages from CD patients to live bacteria, particularly Mycobacterium avium subsp. paratuberculosis (MAP), has not been addressed. Considering MAP has long been associated to CD, our objective was to assess whether macrophages from CD patients showed impaired inflammatory response to infection by MAP comparing to M. avium subsp. avium (MA) and other live intestinal commensal bacteria. Human peripheral blood monocyte-derived macrophages were obtained from CD patients, ulcerative colitis (UC) patients and controls. Following in vitro infection with MAP, MA, Escherichia coli or Enterococcus faecalis, cytokine levels and cell surface receptor expression were evaluated at different time points. Macrophages from CD patients showed impaired TNF-α secretion in response to bacterial challenge, but augmented IL-23 secretion and preserved IL-12 secretion and CD-40 expression. In addition, CD macrophages showed low IL-10 secretion. Macrophages from IBD patients showed increased expression of TLR-2 and -4, unaffected by infection. Differences in cytokine secretion observed after bacterial challenge were not MAP-specific, as other bacteria (E. coli and MA) showed similar effects. Macrophages from UC patients showed a less compromised TNF-α synthesis in response to mycobacterial infection than CD macrophages, with increased constitutive IL-12 secretion, and preserved IL-10 secretion. The increased IL-23 levels in response to infection and decreased IL-10 production observed in macrophages from CD patients may contribute to the inflammatory exacerbation observed in those patients.

Increased viability but decreased culturability of Mycobacterium avium subsp. paratuberculosis in macrophages from inflammatory bowel disease patients under Infliximab treatment.

Mycobacterium avium subsp. paratuberculosis (MAP) has long been implicated as a triggering agent in Crohn’s disease (CD). In this study, we investigated the growth/persistence of both M. avium subsp. hominissuis (MAH) and MAP, in macrophages from healthy controls (HC), CD and ulcerative colitis patients. For viability assessment, both CFU counts and a pre16SrRNA RNA/DNA ratio assay (for MAP) were used. Phagolysosome fusion was evaluated by immunofluorescence, through analysis of LAMP-1 colocalization with MAP. IBD macrophages were more permissive to MAP survival than HC macrophages (a finding not evident with MAH), but did not support MAP active growth. The lower MAP CFU counts in macrophage cultures associated with Infliximab treatment were not due to increased killing, but possibly to elevation in the proportion of intracellular dormant non-culturable MAP forms, as MAP showed higher viability in those macrophages. Increased MAP viability was not related to lack of phagolysosome maturation. The predominant induction of MAP dormant forms by Infliximab treatment may explain the lack of MAP reactivation during anti-TNF therapy of CD but does not exclude the possibility of MAP recrudescence after termination of therapy.

Editorial: Understanding Crohn’s Disease: Immunity, Genes and Microbes

Crohns disease (CD) is a chronic debilitating syndrome, associated with considerable morbidity and resulting in elevated public health costs every year. CD and ulcerative colitis (UC) are the two major forms of inflammatory bowel disease (IBD), which has long been characterized as an exacerbated inflammatory response to common antigenic stimuli in the gut due to immune dysregulation. Although both CD and UC share some common patterns, they are distinct diseases. In fact, while UC is characterized by a diffuse mucosal inflammation involving mainly the rectum and adjacent colonic tissue, CD is a transmural inflammatory disease that may involve any part of the gastrointestinal tract, from the mouth to the anus, although in most cases the terminal ileum is affected. Etiology of IBD, particularly CD, has been long debated and is likely to involve the contribution of multiple factors, making its study challenging. Among those contributing factors are genetic inheritance, epigenetic mechanisms, infection with particular pathobionts, and the gut microbiota in general. This topic aimed at bringing together contributions covering aspects related primarily to CD etiol-ogy and immunopathology, helping to drive forward a more comprehensive understanding of this challenging syndrome. Genetic inheritance is an important predisposing factor for IBD. In a comprehensive review, Loddo and Romano point out the importance of genetic traits in susceptibility to IBD. The authors discuss the success of next-generation sequencing in the investigation of rare monogenic susceptibility variants, implicated in early-onset and very early-onset IBD. They also address the importance of epigenetic mechanisms, such as DNA methylation, in linking environmental stress and gene expression and discuss the use of microRNAs as biomarkers and therapeutical targets in IBD. The first genetic variant conferring susceptibility to ileal CD was located in the nucleotide oligomerization domain 2 (NOD2) gene (1), a gene implicated in recognition of bacterial muramyl dipeptide. Sidiq et al. highlight the role played by epithelial cell NOD2 expression in maintaining gut homeostasis and regulating ileal microbiota composition and address the consequences of a deficient NOD2 variant in gut dysregulation. As described in an original research article, Parkhouse and Monie tested whether loss-of-function NOD2 variants conferring susceptibility to CD (R702W, G908R, and L1007fsincC) exhibited deficient binding to receptor-interacting protein kinase 2, an adaptor protein implicated in NOD2 signaling. They found that impairment of NOD2 signaling shown by variants containing CD-susceptibility polymorphisms did not correlate with deficient RIP2 binding, concluding that the causes for impairment are multifactorial. A …