Browsing by Author "Brombacher, Frank"
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- ItemOpen AccessArteannuin-B and (3-Chlorophenyl)-2-Spiroisoxazoline Derivative Exhibit Anti-Inflammatory Effects in LPS-Activated RAW 264.7 Macrophages and BALB/c Mice-Induced Proinflammatory Responses via Downregulation of NF-κB/P38 MAPK Signaling(2022-11-20) Sawhney, Gifty; Rasool, Javeed Ur; Saroch, Diksha; Ozturk, Mumin; Brombacher, Frank; Ahmad, Bilal; Bhagat, Asha; Ali, Asif; Parihar, Suraj P.; Ahmed, ZabeerHost inflammatory responses are key to protection against injury; however, persistent inflammation is detrimental and contributes to morbidity and mortality. Herein, we demonstrated the anti-inflammatory role of Arteannuin-B (1) and its new spirocyclic-2-isoxazoline derivative JR-9 and their side effects in acute inflammatory condition in vivo using LPS-induced cytokines assay, carrageenan-induced paw edema, acetic acid-induced writhing and tail immersion. The results show that the spirocyclic-2-isoxazoline derivative is a potent anti-inflammatory agent with minimal cell toxicity as compared to Arteannuin-B. In addition, the efficacies of these compounds were also validated by flow cytometric, computational, and histopathological analysis. Our results show that the anti-inflammatory response of JR-9 significantly reduces the ability of mouse macrophages to produce NO, TNF-α, and IL-6 following LPS stimulation. Therefore, JR-9 is a prospective candidate for the development of anti-inflammatory drugs and its molecular mechanism is likely related to the regulation of NF-κB and MAPK signaling pathway.
- ItemOpen AccessBALB/c mice deficient in CD4 T cell IL-4Rα expression control Leishmania mexicana Load although female but not male mice develop a healer phenotype(Public Library of Science, 2011) Bryson, Karen J; Millington, Owain R; Mokgethi, Thabang; McGachy, H Adrienne; Brombacher, Frank; Alexander, JamesImmunologically intact BALB/c mice infected with Leishmania mexicana develop non-healing progressively growing lesions associated with a biased Th2 response while similarly infected IL-4Rα-deficient mice fail to develop lesions and develop a robust Th1 response. In order to determine the functional target(s) for IL-4/IL-13 inducing non-healing disease, the course of L. mexicana infection was monitored in mice lacking IL-4Rα expression in specific cellular compartments. A deficiency of IL-4Rα expression on macrophages/neutrophils (in LysMcreIL-4Rα−/lox animals) had minimal effect on the outcome of L. mexicana infection compared with control (IL-4Rα−/flox) mice. In contrast, CD4+ T cell specific (LckcreIL-4Rα−/lox) IL-4Rα−/− mice infected with L. mexicana developed small lesions, which subsequently healed in female mice, but persisted in adult male mice. While a strong Th1 response was manifest in both male and female CD4+ T cell specific IL-4Rα−/− mice infected with L. mexicana, induction of IL-4 was manifest in males but not females, independently of CD4+ T cell IL-4 responsiveness. Similar results were obtained using pan-T cell specific (iLckcreIL-4Rα−/lox) IL-4Rα−/− mice. Collectively these data demonstrate that upon infection with L. mexicana, initial lesion growth in BALB/c mice is dependent on non-T cell population(s) responsive to IL-4/IL-13 while progressive infection is dependent on CD4+ T cells responsive to IL-4.
- ItemOpen AccessA cross-reactive monoclonal antibody to nematode haemoglobin enhances protective immune responses to Nippostrongylus brasiliensis(Public Library of Science, 2013) Nieuwenhuizen, Natalie E; Meter, Jeanne M; Horsnell, William G; Hoving, J Claire; Fick, Lizette; Sharp, Michael F; Darby, Matthew G; Parihar, Suraj P; Brombacher, Frank; Lopata, Andreas LBackground: Nematode secreted haemoglobins have unusually high affinity for oxygen and possess nitric oxide deoxygenase, and catalase activity thought to be important in protection against host immune responses to infection. In this study, we generated a monoclonal antibody (48Eg) against haemoglobin of the nematode Anisakis pegreffii, and aimed to characterize cross-reactivity of 4E8g against haemoglobins of different nematodes and its potential to mediate protective immunity against a murine hookworm infection. Methodology/Principal Findings: Immunoprecipitation was used to isolate the 4E8g-binding antigen in Anisakis and Ascaris extracts, which were identified as haemoglobins by peptide mass fingerprinting and MS/MS. Immunological cross-reactivity was also demonstrated with haemoglobin of the rodent hookworm N. brasiliensis. Immunogenicity of nematode haemoglobin in mice and humans was tested by immunoblotting. Anisakis haemoglobin was recognized by IgG and IgE antibodies of Anisakis-infected mice, while Ascaris haemoglobin was recognized by IgG but not IgE antibodies in mouse and human sera. Sequencing of Anisakis haemoglobin revealed high similarity to haemoglobin of a related marine nematode, Psuedoterranova decipiens, which lacks the four –HKEE repeats of Ascaris haemoglobin important in octamer assembly. The localization of haemoglobin in the different parasites was examined by immunohistochemistry and associated with the excretory-secretary ducts in Anisakis, Ascaris and N. brasiliensis. Anisakis haemoglobin was strongly expressed in the L3 stage, unlike Ascaris haemoglobin, which is reportedly mainly expressed in adult worms. Passive immunization of mice with 4E8g prior to infection with N. brasiliensis enhanced protective Th2 immunity and led to a significant decrease in worm burdens. Conclusion: The monoclonal antibody 4E8g targets haemoglobin in broadly equivalent anatomical locations in parasitic nematodes and enhances host immunity to a hookworm infection.
- ItemOpen AccessDelayed goblet cell hyperplasia, acetylcholine receptor expression, and worm expulsion in SMC-specific IL-4Ralpha-deficient mice(Public Library of Science, 2007) Horsnell, William G C; Cutler, Antony J; Hoving, J Claire; Mearns, Helen; Myburgh, Elmarie; Arendse, Berenice; Finkelman, Fred D; Owens, Gary K; Erle, Dave; Brombacher, FrankInterleukin 4 receptor α (IL-4Rα) is essential for effective clearance of gastrointestinal nematode infections. Smooth muscle cells are considered to play a role in the type 2 immune response-driven expulsion of gastrointestinal nematodes. Previous studies have shown in vitro that signal transducer and activator of transcription 6 signaling in response to parasitic nematode infection significantly increases smooth muscle cell contractility. Inhibition of the IL-4Rα pathway inhibits this response. How this response manifests itself in vivo is unknown. In this study, smooth muscle cell IL-4Rα-deficient mice (SM-MHC Cre IL-4Rα −/lox ) were generated and characterized to uncover any role for IL-4/IL-13 in this non-immune cell type in response to Nippostrongylus brasiliensis infection. IL-4Rα was absent from α-actin-positive smooth muscle cells, while other cell types showed normal IL-4Rα expression, thus demonstrating efficient cell-type-specific deletion of the IL-4Rα gene. N. brasiliensis -infected SM-MHC Cre IL-4Rα −/lox mice showed delayed ability to resolve infection with significantly prolonged fecal egg recovery and delayed worm expulsion. The delayed expulsion was related to a delayed intestinal goblet cell hyperplasia, reduced T helper 2 cytokine production in the mesenteric lymph node, and reduced M3 muscarinic receptor expression during infection. Together, these results demonstrate that in vivo IL-4Rα-responsive smooth muscle cells are beneficial for N. brasiliensis expulsion by coordinating T helper 2 cytokine responses, goblet hyperplasia, and acetylcholine responsiveness, which drive smooth muscle cell contractions.
- ItemOpen AccessDeletion of IL-4 receptor alpha on dendritic cells renders BALB/c mice hypersusceptible to Leishmania major infection(2013) Hurdayal, Ramona; Brombacher, Frank; Revaz-Breton, MelanieIn BALB/c mice, susceptibility to infection with the intracellular parasite Leishmania major is driven largely by the development of T helper 2 (Th2) responses and the production of interleukin (IL)-4 and IL-13, which share a common receptor subunit, the IL-4 receptor alpha chain (IL-4α ). While IL-4 is the main inducer of Th2 responses, paradoxically it has been shown that exogenously administered IL-4 can promote dendritic cell IL-12 production and enhance Th1 development if given early during infection.
- ItemOpen AccessDeletion of IL-4 receptor alpha on dendritic cells renders BALB/c mice hypersusceptible to Leishmania major infection(Public Library of Science, 2013) Hurdayal, Ramona; Nieuwenhuizen, Natalie E; Revaz-Breton, Mélanie; Smith, Liezel; Hoving, Jennifer C; Parihar, Suraj P; Reizis, Boris; Brombacher, FrankIn BALB/c mice, susceptibility to infection with the intracellular parasite Leishmania major is driven largely by the development of T helper 2 (Th2) responses and the production of interleukin (IL)-4 and IL-13, which share a common receptor subunit, the IL-4 receptor alpha chain (IL-4Rα). While IL-4 is the main inducer of Th2 responses, paradoxically, it has been shown that exogenously administered IL-4 can promote dendritic cell (DC) IL-12 production and enhance Th1 development if given early during infection. To further investigate the relevance of biological quantities of IL-4 acting on DCs during in vivo infection, DC specific IL-4Rα deficient (CD11ccreIL-4Rα-/lox) BALB/c mice were generated by gene targeting and site-specific recombination using the cre/loxP system under control of the cd11c locus. DNA, protein, and functional characterization showed abrogated IL-4Rα expression on dendritic cells and alveolar macrophages in CD11ccreIL-4Rα-/lox mice. Following infection with L. major, CD11ccreIL-4Rα-/lox mice became hypersusceptible to disease, presenting earlier and increased footpad swelling, necrosis and parasite burdens, upregulated Th2 cytokine responses and increased type 2 antibody production as well as impaired classical activation of macrophages. Hypersusceptibility in CD11ccreIL-4Rα-/lox mice was accompanied by a striking increase in parasite burdens in peripheral organs such as the spleen, liver, and even the brain. DCs showed increased parasite loads in CD11ccreIL-4Rα-/lox mice and reduced iNOS production. IL-4Rα-deficient DCs produced reduced IL-12 but increased IL-10 due to impaired DC instruction, with increased mRNA expression of IL-23p19 and activin A, cytokines previously implicated in promoting Th2 responses. Together, these data demonstrate that abrogation of IL-4Rα signaling on DCs is severely detrimental to the host, leading to rapid disease progression, and increased survival of parasites in infected DCs due to reduced killing effector functions.
- ItemOpen AccessDendritic cell-mediated vaccination relies on interleukin-4 receptor signaling to avoid tissue damage after Leishmania major infection of BALB/c mice(Public Library of Science, 2012) Masic, Anita; Hurdayal, Ramona; Nieuwenhuizen, Natalie E; Brombacher, Frank; Moll, HeidrunPrevention of tissue damages at the site of Leishmania major inoculation can be achieved if the BALB/c mice are systemically given L. major antigen (LmAg)-loaded bone marrow-derived dendritic cells (DC) that had been exposed to CpG-containing oligodeoxynucleotides (CpG ODN). As previous studies allowed establishing that interleukin-4 (IL-4) is involved in the redirection of the immune response towards a type 1 profile, we were interested in further exploring the role of IL-4. Thus, wild-type (wt) BALB/c mice or DC-specific IL-4 receptor alpha (IL-4Rα)-deficient (CD11ccreIL-4Rα−/lox) BALB/c mice were given either wt or IL-4Rα-deficient LmAg-loaded bone marrow-derived DC exposed or not to CpG ODN prior to inoculation of 2×105 stationary-phase L. major promastigotes into the BALB/c footpad. The results provide evidence that IL4/IL-4Rα-mediated signaling in the vaccinating DC is required to prevent tissue damage at the site of L. major inoculation, as properly conditioned wt DC but not IL-4Rα-deficient DC were able to confer resistance. Furthermore, uncontrolled L. major population size expansion was observed in the footpad and the footpad draining lymph nodes of CD11ccreIL-4Rα−/lox mice immunized with CpG ODN-exposed LmAg-loaded IL-4Rα-deficient DC, indicating the influence of IL-4Rα-mediated signaling in host DC to control parasite replication. In addition, no footpad damage occurred in BALB/c mice that were systemically immunized with LmAg-loaded wt DC doubly exposed to CpG ODN and recombinant IL-4. We discuss these findings and suggest that the IL4/IL4Rα signaling pathway could be a key pathway to trigger when designing vaccines aimed to prevent damaging processes in tissues hosting intracellular microorganisms.
- ItemOpen AccessDetermination of the role of cytokines using gene deficient mice in African trypanosomiasis infection(2008) Barkhuizen, Mark; Brombacher, Frank; Magez, SefanAfrican trypanosomiasis encompasses diseases caused by pathogenic trypanosomes, infecting both humans and animals alike. To determine the immunological role of IL=12 family members in Trypanosoma brucei brucei, Trypanosoma evansi and Trypanosoma congolense infections, IL-12p35¯/¯, IL-12p40¯/¯ and IL-12p35¯/¯/p40¯/¯ mice were used. While the two latter mouse strains lack all IL-12 homologues, IL-12p35¯/¯ mice still produce IL-12p80 homodimers and IL-23. In infection with T.b. brucei and T.evansi; IL-12p35¯/¯, IL-12p40¯/¯ or IL-12p35¯/¯/p40¯/¯ mice were susceptible to both these pathogens, demonstrated by increased mortality compared to wild type C57BL/6 mice. The different IL-12 deficient mouse strains showed similar mortality kinetics, suggesting that IL-12p70 but not the IL-12p80 homodimer or IL-23 plays a crucial role in survival. Similarly, parasitemia control was reduced in the absence of IL-12p70. While plasma levels of IgM and IgG2c were similar between IL-12 deficient mice and wild type mice, IF-γ production. As IFN-γR¯/¯ mice were also highly susceptible to both T.b. brucei and T. evansi, IL-12p70-dependent IFN-γ production seems to be important mechanism involved in resistance against both these pathogens.
- ItemOpen AccessThe function of IL-4Rα expression on key immune cells during experimental Nippostrongylus brasiliensis infections(2006) Mearns, Helen; Brombacher, Frank; Horsnell, WilliamThe lifecycle of the parasitic nematode Nippostrogylus brasiliensis resembles that of the human hookworms Necator americanus and Ancylostoma duodenale and as such is a useful murine model for studying hookworm disease.
- ItemOpen AccessFunctional miRNA-based Phenotypic Screening as a tool to delineate HIV-host interactions and facilitate Novel Drug DiscoveryNaidoo, Jerolen; Brombacher, Frank; Mhlanga, Musa M; Barichievy, Samantha; anti-HIV therapiesHuman Immunodeficiency Virus (HIV) is the causative agent of AIDS, a disease which affects over 24 million people globally and for which there is neither curative treatment nor vaccine available. As an intracellular pathogen that encodes only 15 proteins HIV-1 is highly dependent upon its host's cellular machinery in order to complete its life cycle. Host-directed therapy thus represents a potentially lucrative strategy for the development of novel anti-HIV therapies. microRNAs (miRNAs) are short noncoding RNA molecules that function as part of the endogenous RNA interference system which governs post transcriptional gene regulation. Current knowledge has placed miRNAs at the crux of HIV-host interactions, yet the functional relevance of the majority of the human miRNAome with regards to HIV replication has remained unknown. A microscopy-based high content screening (HCS) approach was thus developed to systematically evaluate the significance of augmenting or inhibiting the function of individual host miRNAs on the replication dynamics of HIV. A bespoke image analysis and data mining pipeline recovered 56 host miRNAs associated with suppressed HIV replication and 28 host miRNAs associated with enhanced HIV replication. Notably, the HIV-modulating potential of 80 of these miRNAs was previously unknown. Furthermore, HCS also uncovered a novel role for the miR-200 family in the modulation of HIV replication. In silico miRNA target identification and pathway enrichment analysis identified 24 pathways associated exclusively with suppressed HIV replication, 10 pathways associated exclusively with enhanced HIV replication and 38 functional pathways enriched for both enhanced and suppressed viral replication. These included a number of pathways previously implicated in HIV replication such as the PI3K, MAPK, TNF and WNT signalling pathways but also revealed novel functional associations including that of the Hippo signalling pathway. Intriguingly pathway analysis revealed an enrichment for host factors associated with viral carcinogenesis and a convergence on host processes and functional targets classically associated with chemotherapy including host DNA damage repair, cell cycle and tyrosine kinase receptor-mediated signalling. Experimental validation confirmed that HIV replication induced an aberrant cell survival phenotype in response to chemically induced DNA damage but this effect was reversed when DNA damage was induced prior to HIV exposure. A series of compound-based validation screens were thus undertaken in order to verify the functional associations recovered by miRNA screening. A targeted collection of 293 small molecule inhibitors, including a number of FDA-approved chemotherapeutics, were screened for HIV modulating activity. Novel anti-HIV activity was recovered for over 40 compounds including a number of FDA-approved therapies. Compound-target enrichment analysis revealed a strong concordance with functional associations initially described by miRNA-based HCS including EGFR-mediated signalling and DNA damage repair. Concordant HIV-suppressive activity was also recovered for miRNAs and compounds with common functional targets. The outcomes of this study thus represent a significant and novel contribution to current knowledge on HIV-host interactions. Furthermore, these findings have characterised novel miRNA and small molecule candidates for the treatment of HIV and have successfully demonstrated the utility of miRNA-based HCS for novel-drug discovery and drug repositioning.
- ItemOpen AccessGenome-wide profiling of transcribed enhancers during macrophage activation(BioMed Central, 2017-10-23) Denisenko, Elena; Guler, Reto; Mhlanga, Musa M; Suzuki, Harukazu; Brombacher, Frank; Schmeier, SebastianBackground: Macrophages are sentinel cells essential for tissue homeostasis and host defence. Owing to their plasticity, macrophages acquire a range of functional phenotypes in response to microenvironmental stimuli, of which M(IFN-γ) and M(IL-4/IL-13) are well known for their opposing pro- and anti-inflammatory roles. Enhancers have emerged as regulatory DNA elements crucial for transcriptional activation of gene expression. Results: Using cap analysis of gene expression and epigenetic data, we identify on large-scale transcribed enhancers in bone marrow-derived mouse macrophages, their time kinetics, and target protein-coding genes. We observe an increase in target gene expression, concomitant with increasing numbers of associated enhancers, and find that genes associated with many enhancers show a shift towards stronger enrichment for macrophage-specific biological processes. We infer enhancers that drive transcriptional responses of genes upon M(IFN-γ) and M(IL-4/IL-13) macrophage activation and demonstrate stimuli specificity of regulatory associations. Finally, we show that enhancer regions are enriched for binding sites of inflammation-related transcription factors, suggesting a link between stimuli response and enhancer transcriptional control. Conclusions: Our study provides new insights into genome-wide enhancer-mediated transcriptional control of macrophage genes, including those implicated in macrophage activation, and offers a detailed genome-wide catalogue of transcribed enhancers in bone marrow-derived mouse macrophages.
- ItemOpen AccessHigh throughput RNA interference based screen for the identification of genes implicated in listeria monocytogenes infection(2014) Tantoh, Asongwe Lionel Ateh; Brombacher, Frank; Mhlanga, N Emans MThe facultative intracellular pathogen L. monocytogenes has acquired a versatile arsenal of adaptor proteins. It uses these proteins to invade and undermine the host immune surveillance system, as well as to survive and propagate within the host cell. Our understanding of how L. monocytogenes infects cells points towards an interplay between bacterial proteins and human host factors. It has become apparent that this interplay is critical for the establishment and spread of infection. Here we describe a high throughput screening approach to assay for potential human host factors involved in L. monocytogenes infection. In this methodology, we applied a high-density siRNA array comprising of the kinome, phosphatome, ubiquitome and protease encoding human genes that enabled rapid parallel screening for human host factors through reverse transfection into overlaid HeLa cells. The siRNA transfected HeLa cells were exposed to L. monocytogenes expressing the green fluorescent protein. The high content screen comprised a total of 15 replicate screens or 47, 250 individual siRNA experiments. Using a combination of image-based analysis and the data-mining tool of Principal Component Analysis, genes whose silencing visually impaired or enhanced bacteria invasion/proliferation were selected as candidate genes. The 65 strongest genes were subjected to a secondary screening for validation. Among the 15 confirmed hits, we recognized some cellular membrane proteins that might function as cell receptors for bacteria entry and others that may be related to calcium release triggered by bacteria during cell invasion. In addition, we identified a transcription factor bromo adjacent homology domain-containing 1(BAHD1) protein, whose interplay with the bacterial virulence factor LntA has recently been shown to modulate IFN-λ-mediated immune response to control bacterial colonization of the host cell. Also, we identified the inositol polyphosphate-5-phosphatase (INPP5B) that may regulate bacterial internalization by modulating actin dynamics at bacterial internalization sites as shown with oculocerebrorenal syndrome of Lowe (OCRL), whose OCRL gene shares an identical sequence and overlapping enzyme activities with INPP5B. We also identified the large tumor suppressor kinase 2 (LATS2) that may play a potential role in bacterial colonization by modulating the expression of the host E-cad receptor gene. This study represents a significant step toward unveiling the key molecular requirements for host cell invasion and undoubtedly provides an avenue of new therapeutic drugs against L. monocytogenes infection.
- ItemOpen AccessIdentification of genes involved in macrophage activation and effector functions against intracellular pathogens(2006) Schwegmann, Anita Ruth; Brombacher, FrankThis dissertation addressed the hypothesis that macrophages have an alternative killing mechanism that is independent of superoxide and nitric oxide but dependent on IFN-γ, TNF and C/EBPβ. Since the mechanism and the genes involved in this alternative pathway are mostly unknown, the aim of this dissertation was to identify these macrophage effector genes and to functionally characterize their role during infection utilizing gene deficient mouse models. Since mice deficient for C/EBPβ (C/EBPβ-/-) expressed normal levels of IFN-y and TNF during Listeria monocytogenes infection, the macrophage effector genes involved in confinement and killing of L. monocytogenes were postulated to be downstream of C/EBPβ. Furthermore, C/EBPβ-/- mice are highly susceptible L. monocytogenes due to impaired listericidal activity. Comparison of the gene expression profiles of WT and C/EBPβ-/- macrophages infected with L. monocytogenes was postulated to increase the probability of identifying these effector genes, which would be differentially expressed between the two groups. Comparative gene expression profiling by DNA microarrays between L. monocytogenes in infected WT and C/EBPβ-/- macrophages, successfully identified 1268 genes to be differentially expressed between the two groups. A focussed functional clustering strategy reduced the number of candidate genes to 220. PKCδ was selected for further study since it was involved in humoral defense, immune signalling, production of superoxide, regulation of transcription and may be putatively transcriptionally regulated by C/EBPβ. Furthermore, PKCδ was indirectly shown to promote L. monocytogenes escape from the phagosome and to negatively regulate transcription activity of C/EBPβ. In addition, since PKCδ was un-regulated, as shown by microarray and confirmed by RT-PCR, in L. monocytogenes infected C/EBPβ-/- macrophages, it was therefore thought to play a detrimental role during L. monocytogenes. However, since this premise has never been investigated directly, the role PKCδ during innate immunity against L monocytogenes was examined using the PKCδ deficient (PKCδ-/-) mouse model. Data in this dissertation provides new insight into the role of PKCδ during innate immunity to L. monocytogenes. PKCδ-/- mice were highly susceptible to L. monocytogenes due to enhanced listerial escape and impaired listericidal activity. Despite full macrophage activation and production of nitric oxide, PKCδ-/- mice displayed uncontrolled bacterial growth and dissemination of L. monocytogenes, which led to early death of the mice. In contrast, PKCδ-/- mice were able to control Mycobacterium infection as well as WT mice, suggesting that the activity of PKCδ may be negatively regulated by L. monocytogenes. A systems biology approach generated the hypothesis that PKCδ may promote Rab5a activation, which together with localized release of superoxide into the phagosome and activation of C/EBPβ by PKCδ, resulted in the confinement of the L. monocytogenes within the phagosome. Alternatively, PKCδ may act in a separate pathway that confines L monocytogenes within the phagosome, by activating and/or synergizing with unidentified proteins to neutralize that activity of listerial LLO and PI-PLC. Data in this dissertation clearly demonstrates that PKCδ is critical for confinement of L monocytogenes within phagosomes and may be part of a listericidal mechanism that is independent or nitric oxide, superoxide and pro-inflammatory cytokines.
- ItemOpen AccessIL-4 receptor-alpha-dependent control of Cryptococcus neoformans in the early phase of pulmonary infection(Public Library of Science, 2014) Grahnert, Andreas; Richter, Tina; Piehler, Daniel; Eschke, Maria; Schulze, Bianca; Müller, Uwe; Protschka, Martina; Köhler, Gabriele; Sabat, Robert; Brombacher, Frank; Alber, GottfriedCryptococcus neoformans is an opportunistic fungal pathogen that causes lung inflammation and meningoencephalitis in immunocompromised people. Previously we showed that mice succumb to intranasal infection by induction of pulmonary interleukin (IL)-4Rα-dependent type 2 immune responses, whereas IL-12-dependent type 1 responses confer resistance. In the experiments presented here, IL-4Rα −/− mice unexpectedly show decreased fungal control early upon infection with C. neoformans , whereas wild-type mice are able to control fungal growth accompanied by enhanced macrophage and dendritic cell recruitment to the site of infection. Lower pulmonary recruitment of macrophages and dendritic cells in IL-4Rα −/− mice is associated with reduced pulmonary expression of CCL2 and CCL20 chemokines. Moreover, IFN-γ and nitric oxide production are diminished in IL-4Rα −/− mice compared to wild-type mice. To directly study the potential mechanism(s) responsible for reduced production of IFN-γ, conventional dendritic cells were stimulated with C. neoformans in the presence of IL-4 which results in increased IL-12 production and reduced IL-10 production. Together, a beneficial role of early IL-4Rα signaling is demonstrated in pulmonary cryptococcosis, which contrasts with the well-known IL-4Rα-mediated detrimental effects in the late phase.
- ItemOpen AccessIL-4/IL-13 independent goblet cell hyperplasia in experimental helminth infections(BioMed Central Ltd, 2008) Marillier, Reece; Michels, Chesney; Smith, Elizabeth; Fick, Lizette; Leeto, Mosiuoa; Dewals, Benjamin; Horsnell, William; Brombacher, FrankBACKGROUND: Intestinal mucus production by hyperplasic goblet cells is a striking pathological feature of many parasitic helminth infections and is related to intestinal protection and worm expulsion. Induction of goblet cell hyperplasia is associated with TH2 immune responses, which in helminth infections are controlled primarily by IL-13, and also IL-4. In the study presented here we examine the goblet cell hyperplasic response to three experimental parasitic helminth infections; namely Nippostrongylus brasiliensis, Syphacia obvelata and Schistosoma mansoni. RESULTS: As expected N. brasiliensis infection induced a strong goblet cell hyperplasia dependent on IL-4/IL-13/IL-4Ralpha expression. In contrast, and despite previously published transiently elevated IL-4/IL-13 levels, S. obvelata infections did not increase goblet cell hyperplasia in the colon. Furthermore, induction of goblet cell hyperplasia in response to S. mansoni eggs traversing the intestine was equivalent between BALB/c, IL-4/IL-13-/- and IL-4Ralpha-/- mice. CONCLUSION: Together these data demonstrate that intestinal goblet cell hyperplasia can be independent of TH2 immune responses associated with parasitic helminth infections.
- ItemOpen AccessIL-4/IL-13-inducible lincRNA-MIR99AHG regulates macrophage polarization and promotes intracellular survival of Mycobacterium tuberculosis(2020) Gcanga, Lona; Guler, Reto; Tamgue, Ousman; Brombacher, FrankTuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) kills 1.6 million people worldwide every year, and there is an urgent need for targeting host-pathogen interactions as a strategy to reduce mycobacterial resistance to current antimicrobials. Non-coding RNAs are emerging as important regulators of numerous biological processes and avenues for exploitation in host-directed therapeutics. Although long non-coding RNAs (lncRNAs) are abundantly expressed in immune cells, their functional role in gene regulation and bacterial infections remains under-studied. Here, we identify an immunoregulatory, lincRNA-MIR99AHG, which is upregulated in macrophages upon IL-4/IL-13 stimulation and downregulated after Mtb infection and in active TB patients. To evaluate the functional role of lincRNA-MIR99AHG, we employed antisense GapmeR-mediated lncRNA knockdown experiments. Knockdown of lincRNA-MIR99AHG with LNA-GapmeRs significantly reduced intracellular Mtb growth in mouse and human macrophages and reduced proinflammatory cytokine production. In addition, in vivo treatment with MIR99AHG LNA-GapmeRs reduced the mycobacterial burden in the lung and spleen. In vivo LNA-GapmeR treatment experiments demonstrated a role of lincRNA-MIR99AHG as a regulator of macrophage polarization and a host-mediated response post Mtb infection. Further, lincRNA-MIR99AHG translocated to the nucleus and interacts with a high affinity to hnRNPA2/B1 following IL-4/IL-13 stimulation and Mtb infection. Together, these findings identify lincRNA-MIR99AHG as a positive regulator of inflammation to promote Mtb growth and a possible for host-directed targeting or for adjunctive therapeutics against TB.
- ItemOpen AccessIL-4Rα-Dependent Alternative Activation of Macrophages Is Not Decisive for Mycobacterium tuberculosis Pathology and Bacterial Burden in Mice(Public Library of Science, 2015) Guler, Reto; Parihar, Suraj P; Savvi, Suzana; Logan, Erin; Schwegmann, Anita; Roy, Sugata; Nieuwenhuizen, Natalie E; Ozturk, Mumin; Schmeier, Sebastian; Suzuki, Harukazu; Brombacher, FrankClassical activation of macrophages (caMph or M1) is crucial for host protection against Mycobacterium tuberculosis ( Mtb ) infection. Evidence suggests that IL-4/IL-13 alternatively activated macrophages (aaMph or M2) are exploited by Mtb to divert microbicidal functions of caMph. To define the functions of M2 macrophages during tuberculosis (TB), we infected mice deficient for IL-4 receptor α on macrophages (LysM cre IL-4Rα -/lox ) with Mtb . We show that absence of IL-4Rα on macrophages does not play a major role during infection with Mtb H37Rv, or the clinical Beijing strain HN878. This was demonstrated by similar mortality, bacterial burden, histopathology and T cell proliferation between infected wild-type (WT) and LysM cre IL-4Rα -/lox mice. Interestingly, we observed no differences in the lung expression of inducible nitric oxide synthase (iNOS) and Arginase 1 (Arg1), well-established markers for M1/M2 macrophages among the Mtb -infected groups. Kinetic expression studies of IL-4/IL-13 activated bone marrow-derived macrophages (BMDM) infected with HN878, followed by gene set enrichment analysis, revealed that the MyD88 and IL-6, IL-10, G-CSF pathways are significantly enriched, but not the IL-4Rα driven pathway. Together, these results suggest that IL-4Rα-macrophages do not play a central role in TB disease progression.
- ItemOpen AccessIL-4Rα-independent expression of mannose receptor and Ym1 by macrophages depends on their IL-10 responsiveness(Public Library of Science, 2010) Dewals, Benjamin G; Marillier, Reece G; Hoving, Jennifer C; Leeto, Mosiuoa; Schwegmann, Anita; Brombacher, FrankIL-4Rα-dependent responses are essential for granuloma formation and host survival during acute schistosomiasis. Previously, we demonstrated that mice deficient for macrophage-specific IL-4Rα (LysMcreIl4ra−/lox) developed increased hepatotoxicity and gut inflammation; whereas inflammation was restricted to the liver of mice lacking T cell-specific IL-4Rα expression (iLckcreIl4ra−/lox). In the study presented here we further investigated their role in liver granulomatous inflammation. Frequencies and numbers of macrophage, lymphocyte or granulocyte populations, as well as Th1/Th2 cytokine responses were similar in Schistosoma mansoni-infected LysMcreIl4ra−/lox liver granulomas, when compared to Il4ra−/lox control mice. In contrast, a shift to Th1 responses with high IFN-γ and low IL-4, IL-10 and IL-13 was observed in the severely disrupted granulomas of iLckcreIl4ra−/lox and Il4ra−/− mice. As expected, alternative macrophage activation was reduced in both LysMcreIl4ra−/lox and iLckcreIl4ra−/lox granulomas with low arginase 1 and heightened nitric oxide synthase RNA expression in granuloma macrophages of both mouse strains. Interestingly, a discrete subpopulation of SSChighCD11b+I-A/I-EhighCD204+ macrophages retained expression of mannose receptor (MMR) and Ym1 in LysMcreIl4ra−/lox but not in iLckcreIl4ra−/lox granulomas. While aaMφ were in close proximity to the parasite eggs in Il4ra−/lox control mice, MMR+Ym1+ macrophages in LysMcreIl4ra−/lox mice were restricted to the periphery of the granuloma, indicating that they might have different functions. In vivo IL-10 neutralisation resulted in the disappearance of MMR+Ym1+ macrophages in LysMcreIl4ra−/lox mice. Together, these results show that IL-4Rα-responsive T cells are essential to drive alternative macrophage activation and to control granulomatous inflammation in the liver. The data further suggest that in the absence of macrophage-specific IL-4Rα signalling, IL-10 is able to drive mannose receptor- and Ym1-positive macrophages, associated with control of hepatic granulomatous inflammation.
- ItemOpen AccessImmune and allergic responses to Anisakis pegreffii, with focus on the roles of IL-4, IL-13 and the IL-4 receptor alpha(2007) Nieuwenhuizen, Natalie; Lopata, Andreas; Brombacher, FrankThe fish-parasitizing nematode Anisakis pegreffii induces gastrointestinal disease and allergy when ingested by humans, and can cause occupational allergy in seafood processing workers. The present study examines immune and allergic responses to A. pegreffii in wildtype and gene deficient mice, with special focus on interleukin(IL)-4, IL-13, and the IL-4 receptor alpha (IL-4Rα). Experimental murine models of Anisakis infection, Anisakis-induced anaphylaxis and Anisakis-induced dermatitis were established in order to gain insight into the immune responses generated against Anisakis and unravel mechanisms of allergic disease.
- ItemOpen AccessImpact of the parasitic helminth Schistosoma mansoni on host anti-viral vaccine responses: proof of concept from the anti-polio vaccine(2022) Musaigwa, Fungai; Nono, Komguep Justin; Brombacher, FrankSchistosomiasis is a devastating and neglected tropical disease caused by Schistosoma spp. parasites. The disease remains greatly neglected by global health control programmes thus classified as a top neglected tropical disease of humankind. One of the most common species of these parasites, Schistosoma mansoni, causes hepatosplenic schistosomiasis and distributes widely in sub-Saharan Africa. Schistosoma mansoni is physiologically debilitating and potentially deadly with a known potential as a master regulator of the host immune responses. However, the mechanistic bases and the scope of this immunoregulatory potential of S. mansoni still remain poorly understood. This study explored the influence of S. mansoni-driven schistosomiasis on vaccine-induced memory immunity in schoolchildren from a rural endemic area of Cameroon and in laboratory mice. As a proof of concept, a well-established anti-viral vaccination programme with wide global coverage, i.e., anti-polio vaccination, was evaluated for its sustainability in the face of schistosomiasis in human and mouse hosts. Our findings using the poliovirus specific enzyme-linked immunosorbent assay revealed a schistosomiasis-associated impairment of polio specific serologic antibody memory responses in previously vaccinated schoolchildren and mice, as judged by significantly reduced serum anti-polio IgG antibody levels. To explore our findings further, cellular evaluations were conducted using flow cytometry in mouse modes of vaccination and schistosomiasis. The reduction of anti-polio elicited antibody responses was paralleled by the general depletion, through reduced survival and increased cell death, of bone marrow plasma cells and plasma blasts in schistosomiasis-diseased mice. Notably, schistosomiasis reduced memory T cell responses as well in vaccinated hosts and considerably impaired the expression of survival markers on antibody-producing long term plasma cells in the bone marrow. When attempting to control the disease by administration of the sole commercially available drug for schistosomiasis control, praziquantel, the parasite-driven depletion of the plasma cell and memory T cell compartment was restored followed by a partial recovery of antibody-producing abilities in vaccinated hosts. Taken together, our findings unprecedentedly demonstrate how schistosomiasis might negatively influence the efficacy and potentially the effectiveness of anti-viral vaccine memory immunity. Additionally, we present findings on how strategic therapeutic interventions against schistosomiasis might positively influence vaccine-elicited anti-viral immunity in schistosomiasis-diseased hosts. Our results have robust implications for future more informed deployment of effective vaccination campaigns, beyond the sole consideration of coverage and encourage frequent mass drug administration of praziquantel in schistosomiasis-endemic areas to ensure the sustainability of vaccine elicited responses thus protection of the hosts against vaccine preventable diseases.