title: Inflammatory response of TLR4 deficient spleen macrophages (CRL 2471) to Brucella abortus S19 and an isogenic ΔmglA deletion mutant.
abstract: Brucellosis is a worldwide distributed zoonosis caused by members of the genus Brucella. One of them, Brucella abortus, is the etiological agent of bovine brucellosis. With the attenuated strain B. abortus S19 a vaccine is available. However, both, virulence (safety) and the ability to induce a protective B and T cell response (efficacy) have to be tested in suitable assays before successful use in the field. For this purpose, several macrophage cell lines of various origins have been used while splenic macrophages are the preferred host cells in vivo. We here characterized the in vitro response of the murine splenic macrophage cell line CRL 2471(I-13.35) to B. abortus. This cell line still depends on the presence of colony-stimulating factor 1 (CSF1) and is derived from LPS resistant (TLR4 deficient) C3H/HeJ mice. For infection the vaccine strain B. abortus S19A as well as the formerly described isogenic deletion mutant B. abortus S19A ΔmglA 3.14 were used. While numbers of viable bacteria did not differ significantly between the vaccine strain and the deletion mutant at 6h post infection, a higher bacterial load was measured in case of the mutant at 24h and 48h after infection. This was also true, when IFNγ was used for macrophage activation. A comprehensive gene expression profile of macrophages was analysed 6 and 24h after infection by means of an RT-PCR based gene expression array. The mutant strain B. abortus S19A ΔmglA 3.14 elicited a stronger cellular response of the splenic macrophages as compared to the parental vaccine strain. This was most prominent for the pro-inflammatory cytokines IL-1α, IL-1β, TNF-α and IL6 as well as for the chemokine ligands CXCL1, CXCL2, CXCL10, CCL2, CCL5, CCL7, CCL17 and the co-stimulatory molecules CD40 and ICAM1. While these differences were also present in IFNγ-stimulated macrophages, an addition of IFNγ after infection not only resulted in a dramatic increase of the translation of the afore mentioned genes but also resulted in the translation of IFNß1, IL12ß, MIP1α and β (CCL3, CCL4), NOS2 (and SOD2) and FAS.
  
  
  CONCLUSIONS
  The TLR4 deficient murine splenic macrophage cell line CRL 2471 was used for the first time for the characterization of macrophage-Brucella interaction to investigate the pre-immune phase of brucellosis in vitro. Typical pro-inflammatory cytokines and certain surface receptors were differentially induced by B. abortus S19 A and an isogenic ΔmglA deletion mutant in vitro. This model may be useful for further studies to characterize the inflammatory response of splenic macrophages to intracellular gram-negative bacteria avoiding cell responses to soluble LPS.
authors.affiliation: Robert Koch-Institute, Nordufer 20, 13353 Berlin, Germany. Electronic address: jacobj@rki.de.
authors.forename: Jens
authors.initials: J
authors.lastname: Jacob
authors.affiliation: Robert Koch-Institute, Nordufer 20, 13353 Berlin, Germany.
authors.forename: Patricia
authors.initials: P
authors.lastname: Makou
authors.affiliation: Robert Koch-Institute, Nordufer 20, 13353 Berlin, Germany.
authors.forename: Antje
authors.initials: A
authors.lastname: Finke
authors.affiliation: Robert Koch-Institute, Nordufer 20, 13353 Berlin, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Mielke
mesh_terms: D000818:Animals; D002003:Brucella abortus / Q000235:genetics / Q000276:immunology*; D002006:Brucellosis / Q000276:immunology / Q000382:microbiology; D002460:Cell Line; D054414:Chemokine CCL17 / Q000235:genetics / Q000378:metabolism; D018932:Chemokine CCL2 / Q000235:genetics / Q000378:metabolism; D018946:Chemokine CCL5 / Q000235:genetics / Q000378:metabolism; D054410:Chemokine CCL7 / Q000235:genetics / Q000378:metabolism; D054360:Chemokine CXCL1 / Q000235:genetics / Q000378:metabolism; D054357:Chemokine CXCL10 / Q000235:genetics / Q000378:metabolism; D054426:Chemokine CXCL2 / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D054884:Host-Pathogen Interactions / Q000276:immunology; D007371:Interferon-gamma / Q000235:genetics / Q000378:metabolism; D053582:Interleukin-1alpha / Q000235:genetics / Q000378:metabolism; D053583:Interleukin-1beta / Q000235:genetics / Q000378:metabolism; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D016173:Macrophage Colony-Stimulating Factor / Q000737:chemistry; D008264:Macrophages / Q000276:immunology* / Q000382:microbiology; D051379:Mice; D008809:Mice, Inbred C3H; D013154:Spleen / Q000166:cytology* / Q000382:microbiology; D051197:Toll-Like Receptor 4 / Q000235:genetics* / Q000378:metabolism; D059467:Transcriptome; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism
chemical_list: C499538:Ccl17 protein, mouse; C491768:Ccl2 protein, mouse; C503460:Ccl5 protein, mouse; C515451:Ccl7 protein, mouse; D054414:Chemokine CCL17; D018932:Chemokine CCL2; D018946:Chemokine CCL5; D054410:Chemokine CCL7; D054360:Chemokine CXCL1; D054357:Chemokine CXCL10; D054426:Chemokine CXCL2; C475895:Cxcl1 protein, mouse; C485433:Cxcl10 protein, mouse; C404915:Cxcl2 protein, mouse; D053582:Interleukin-1alpha; D053583:Interleukin-1beta; D015850:Interleukin-6; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; D016173:Macrophage Colony-Stimulating Factor; D007371:Interferon-gamma; D007501:Iron
keywords: Brucella abortus; Inflammatory response; Iron; Macrophages; mglA