Frank Brombacher

Batf2/Irf1 Induces Inflammatory Responses in Classically Activated Macrophages, Lipopolysaccharides, and Mycobacterial Infection

Basic leucine zipper transcription factor Batf2 is poorly described, whereas Batf and Batf3 have been shown to play essential roles in dendritic cell, T cell, and B cell development and regulation. Batf2 was drastically induced in IFN-γ–activated classical macrophages (M1) compared with unstimulated or IL-4–activated alternative macrophages (M2). Batf2 knockdown experiments from IFN-γ–activated macrophages and subsequent expression profiling demonstrated important roles for regulation of immune responses, inducing inflammatory and host-protective genes Tnf, Ccl5, and Nos2. Mycobacterium tuberculosis (Beijing strain HN878)–infected macrophages further induced Batf2 and augmented host-protective Batf2-dependent genes, particularly in M1, whose mechanism was suggested to be mediated through both TLR2 and TLR4 by LPS and heat-killed HN878 (HKTB) stimulation experiments. Irf1 binding motif was enriched in the promoters of Batf2-regulated genes. Coimmunoprecipitation study demonstrated Batf2 association with Irf1. Furthermore, Irf1 knockdown showed downregulation of IFN-γ– or LPS/HKTB-activated host-protective genes Tnf, Ccl5, Il12b, and Nos2. Conclusively, Batf2 is an activation marker gene for M1 involved in gene regulation of IFN-γ–activated classical macrophages, as well as LPS/HKTB-induced macrophage stimulation, possibly by Batf2/Irf1 gene induction. Taken together, these results underline the role of Batf2/Irf1 in inducing inflammatory responses in M. tuberculosis infection.

Redefining the transcriptional regulatory dynamics of classically and alternatively activated macrophages by deepCAGE transcriptomics

Classically or alternatively activated macrophages (M1 and M2, respectively) play distinct and important roles for microbiocidal activity, regulation of inflammation and tissue homeostasis. Despite this, their transcriptional regulatory dynamics are poorly understood. Using promoter-level expression profiling by non-biased deepCAGE we have studied the transcriptional dynamics of classically and alternatively activated macrophages. Transcription factor (TF) binding motif activity analysis revealed four motifs, NFKB1_REL_RELA, IRF1,2, IRF7 and TBP that are commonly activated but have distinct activity dynamics in M1 and M2 activation. We observe matching changes in the expression profiles of the corresponding TFs and show that only a restricted set of TFs change expression. There is an overall drastic and transient up-regulation in M1 and a weaker and more sustainable up-regulation in M2. Novel TFs, such as Thap6, Maff, (M1) and Hivep1, Nfil3, Prdm1, (M2) among others, were suggested to be involved in the activation processes. Additionally, 52 (M1) and 67 (M2) novel differentially expressed genes and, for the first time, several differentially expressed long non-coding RNA (lncRNA) transcriptome markers were identified. In conclusion, the finding of novel motifs, TFs and protein-coding and lncRNA genes is an important step forward to fully understand the transcriptional machinery of macrophage activation.

Host-directed drug therapy for tuberculosis

Chemical compounds designed to enhance understanding of host-pathogen interaction together with next-generation smart drugs will rationally drive the discovery of promising new host-directed targets against pathogens including Mycobacterium tuberculosis, the causative agent of tuberculosis.

Targeting Batf2 for infectious diseases and cancer

The family members Batf, Batf2 and Batf3 belong to a class of transcription factors containing basic leucine zipper domains that regulate various immunological functions and control the development and differentiation of immune cells. Functional studies by others demonstrated a predominant role for Batf in controlling Th2 cell functions and lineage development of T lymphocytes as well as a critical role of Batf, Batf2 and Batf3 in CD8α+dendritic cell development. Moreover, Batf family member expression was measured in a vast collection of mouse and human cell types by cap analysis gene expression (CAGE), a recent developed sequencing technology, showing reasonable expression spectrum in immune cells consistent with previously published expression profiles. Batf and Batf3 were highly expressed in lymphocytes and the earlier moderately expressed in myeloid lineages. Batf2 was predominantly expressed in monocytes/macrophages. Functional studies in mice demonstrated that Batf2 has a central role in macrophage activation by regulating inflammatory responses during lipopolysaccharides stimulation and mycobacterial infection. Hence, Batf2 could be used as a biomarker and a potential host directed drug target in tuberculosis. Moreover, Batf2 act as a tumor suppressor gene and augmenting Batf2 in malignant cells might be an encouraging therapeutic treatment against cancer.

IRNdb: the database of immunologically relevant non-coding RNAs

MicroRNAs (miRNAs), long non-coding RNAs (lncRNAs) and other functional non-coding RNAs (ncRNAs) have emerged as pivotal regulators involved in multiple biological processes. Recently, ncRNA control of gene expression has been identified as a critical regulatory mechanism in the immune system. Despite the great efforts made to discover and characterize ncRNAs, the functional role for most remains unknown. To facilitate discoveries in ncRNA regulation of immune system-related processes, we developed the database of immunologically relevant ncRNAs and target genes (IRNdb). We integrated mouse data on predicted and experimentally supported ncRNA-target interactions, ncRNA and gene annotations, biological pathways and processes and experimental data in a uniform format with a user-friendly web interface. The current version of IRNdb documents 12 930 experimentally supported miRNA-target interactions between 724 miRNAs and 2427 immune-related mouse targets. In addition, we recorded 22 453 lncRNA-immune target and 377 PIWI-interacting RNA-immune target interactions. IRNdb is a comprehensive searchable data repository which will be of help in studying the role of ncRNAs in the immune system. Database URL: http://irndb.org

An evaluation of minor groove binders as anti-fungal and anti-mycobacterial therapeutics

This study details the synthesis and biological evaluation of a collection of 19 structurally related Minor Groove Binders (MGBs), derived from the natural product distamycin, which were designed to probe antifungal and antimycobacterial activity. From this initial set, we report several MGBs that are worth more detailed investigation and optimisation. MGB-4, MGB-317 and MGB-325 have promising MIC80s of 2, 4 and 0.25xa0μg/mL, respectively, against the fungus C.xa0neoformans.MGB-353 and MGB-354 have MIC99s of 3.1xa0μM against the mycobacterium M.xa0tuberculosis. The selectivity and activity of these compounds is related to their physicochemical properties and the cell wall/membrane characteristics of the infective agents.

IL-4 Receptor Alpha Signaling through Macrophages Differentially Regulates Liver Fibrosis Progression and Reversal

Chronic hepatitis leads to liver fibrosis and cirrhosis. Cirrhosis is a major cause of worldwide morbidity and mortality. Macrophages play a key role in fibrosis progression and reversal. However, the signals that determine fibrogenic vs fibrolytic macrophage function remain ill defined. We studied the role of interleukin-4 receptor α (IL-4Rα), a potential central switch of macrophage polarization, in liver fibrosis progression and reversal. We demonstrate that inflammatory monocyte infiltration and liver fibrogenesis were suppressed in general IL-4Rα−/− as well as in macrophage-specific IL-4Rα−/− (IL-4RαΔLysM) mice. However, with deletion of IL-4RαΔLysM spontaneous fibrosis reversal was retarded. Results were replicated by pharmacological intervention using IL-4Rα-specific antisense oligonucleotides. Retarded resolution was linked to the loss of M2-type resident macrophages, which secreted MMP-12 through IL-4 and IL-13-mediated phospho-STAT6 activation. We conclude that IL-4Rα signaling regulates macrophage functional polarization in a context-dependent manner. Pharmacological targeting of macrophage polarization therefore requires disease stage-specific treatment strategies. Research in Context Alternative (M2-type) macrophage activation through IL-4Rα promotes liver inflammation and fibrosis progression but speeds up fibrosis reversal. This demonstrates context dependent, opposing roles of M2-type macrophages. During reversal IL-4Rα induces fibrolytic MMPs, especially MMP-12, through STAT6. Liver-specific antisense oligonucleotides efficiently block IL-4Rα expression and attenuate fibrosis progression.

Evaluation of minor groove binders (MGBs) as novel anti-mycobacterial agents, and the effect of using non-ionic surfactant vesicles as a delivery system to improve their efficacy

ObjectivesnThe slow development of major advances in drug discovery for the treatment of Mycobacterium tuberculosis (Mtb) infection suggests a compelling need for evaluation of more effective drug therapies against TB. New classes of drugs are constantly being evaluated for anti-mycobacterial activity with currently a very limited number of new drugs approved for TB treatment. Minor groove binders (MGBs) have previously revealed promising antimicrobial activity against various infectious agents; however, they have not yet been screened against Mtb.nnnMethodsnThe mycobactericidal activity of 96 MGB compounds against Mtb was determined using an H37Rv-GFP microplate assay. MGB hits were screened for their intracellular mycobactericidal efficacy against the clinical Beijing Mtb strain HN878 in bone-marrow-derived macrophages using standard cfu counting. Cell viability was assessed by CellTiter-Blue assays. Selected MGBs were encapsulated into non-ionic surfactant vesicles (NIVs) for drug delivery system evaluation.nnnResultsnH37Rv-GFP screening yielded a hit-list of seven compounds at an MIC99 of between 0.39 and 1.56u2009μM. MGB-362 and MGB-364 displayed intracellular mycobactericidal activity against Mtb HN878 at an MIC50 of 4.09 and 4.19u2009μM, respectively, whilst being non-toxic. Subsequent encapsulation into NIVs demonstrated a 1.6- and 2.1-fold increased intracellular mycobacterial activity, similar to that of rifampicin when compared with MGB-alone formulation.nnnConclusionsnMGB anti-mycobacterial activities together with non-toxic properties indicate that MGB compounds constitute an important new class of drug/chemical entity, which holds promise in future anti-TB therapy. Furthermore, the ability of NIVs to better deliver entrapped MGB compounds to an intracellular Mtb infection suggests further preclinical evaluation is warranted.

Transcriptional landscape of Mycobacterium tuberculosis infection in macrophages

Mycobacterium tuberculosis (Mtb) infection reveals complex and dynamic host-pathogen interactions, leading to host protection or pathogenesis. Using a unique transcriptome technology (CAGE), we investigated the promoter-based transcriptional landscape of IFNγ (M1) or IL-4/IL-13 (M2) stimulated macrophages during Mtb infection in a time-kinetic manner. Mtb infection widely and drastically altered macrophage-specific gene expression, which is far larger than that of M1 or M2 activations. Gene Ontology enrichment analysis for Mtb-induced differentially expressed genes revealed various terms, related to host-protection and inflammation, enriched in up-regulated genes. On the other hand, terms related to dis-regulation of cellular functions were enriched in down-regulated genes. Differential expression analysis revealed known as well as novel transcription factor genes in Mtb infection, many of them significantly down-regulated. IFNγ or IL-4/IL-13 pre-stimulation induce additional differentially expressed genes in Mtb-infected macrophages. Cluster analysis uncovered significant numbers, prolonging their expressional changes. Furthermore, Mtb infection augmented cytokine-mediated M1 and M2 pre-activations. In addition, we identified unique transcriptional features of Mtb-mediated differentially expressed lncRNAs. In summary we provide a comprehensive in depth gene expression/regulation profile in Mtb-infected macrophages, an important step forward for a better understanding of host-pathogen interaction dynamics in Mtb infection.

Chronic schistosomiasis suppresses HIV-specific responses to DNA-MVA and MVA-gp140 Env vaccine regimens despite antihelminthic treatment and increases helminth-associated pathology in a mouse model

Future HIV vaccines are expected to induce effective Th1 cell-mediated and Env-specific antibody responses that are necessary to offer protective immunity to HIV infection. However, HIV infections are highly prevalent in helminth endemic areas. Helminth infections induce polarised Th2 responses that may impair HIV vaccine-generated Th1 responses. In this study, we tested if Schistosoma mansoni (Sm) infection altered immune responses to SAAVI candidate HIV vaccines (DNA and MVA) and an HIV-1 gp140 Env protein vaccine (gp140) and whether parasite elimination by chemotherapy or the presence of Sm eggs (SmE) in the absence of active infection influenced the immunogenicity of these vaccines. In addition, we evaluated helminth-associated pathology in DNA and MVA vaccination groups. Mice were chronically infected with Sm and vaccinated with DNA+MVA in a prime+boost combination or MVA+gp140 in concurrent combination regimens. Some Sm-infected mice were treated with praziquantel (PZQ) prior to vaccinations. Other mice were inoculated with SmE before receiving vaccinations. Unvaccinated mice without Sm infection or SmE inoculation served as controls. HIV responses were evaluated in the blood and spleen while Sm-associated pathology was evaluated in the livers. Sm-infected mice had significantly lower magnitudes of HIV-specific cellular responses after vaccination with DNA+MVA or MVA+gp140 compared to uninfected control mice. Similarly, gp140 Env-specific antibody responses were significantly lower in vaccinated Sm-infected mice compared to controls. Treatment with PZQ partially restored cellular but not humoral immune responses in vaccinated Sm-infected mice. Gp140 Env-specific antibody responses were attenuated in mice that were inoculated with SmE compared to controls. Lastly, Sm-infected mice that were vaccinated with DNA+MVA displayed exacerbated liver pathology as indicated by larger granulomas and increased hepatosplenomegaly when compared with unvaccinated Sm-infected mice. This study shows that chronic schistosomiasis attenuates both HIV-specific T-cell and antibody responses and parasite elimination by chemotherapy may partially restore cellular but not antibody immunity, with additional data suggesting that the presence of SmE retained in the tissues after antihelminthic therapy contributes to lack of full immune restoration. Our data further suggest that helminthiasis may compromise HIV vaccine safety. Overall, these findings suggested a potential negative impact on future HIV vaccinations by helminthiasis in endemic areas.