Guanylate Binding Proteins Enable Rapid Activation of Canonical and Noncanonical Inflammasomes in Chlamydia-Infected Macrophages

Interferon (IFN)-inducible guanylate binding proteins (GBPs) mediate cell-autonomous host resistance to bacterial pathogens and promote inflammasome activation. The prevailing model postulates that these two GBP-controlled activities are directly linked through GBP-dependent vacuolar lysis. It was proposed that the rupture of pathogen-containing vacuoles (PVs) by GBPs destroyed the microbial refuge and simultaneously contaminated the host cell cytosol with microbial activators of inflammasomes. Here, we demonstrate that GBP-mediated host resistance and GBP-mediated inflammatory responses can be uncoupled. We show that PVs formed by the rodent pathogenChlamydia muridarum, so-called inclusions, remain free of GBPs and thatC. muridarumis impervious to GBP-mediated restrictions on bacterial growth. Although GBPs neither bind toC. muridaruminclusions nor restrictC. muridarumgrowth, we find that GBPs promote inflammasome activation inC. muridarum-infected macrophages. We demonstrate thatC. muridaruminfections induce GBP-dependent pyroptosis through both caspase-11-dependent noncanonical and caspase-1-dependent canonical inflammasomes. Among canonical inflammasomes, we find thatC. muridarumand the human pathogenChlamydia trachomatisactivate not only NLRP3 but also AIM2. Our data show that GBPs support fast-kinetics processing and secretion of interleukin-1β (IL-1β) and IL-18 by the NLRP3 inflammasome but are dispensable for the secretion of the same cytokines at later times postinfection. Because IFN-γ fails to induce IL-1β transcription, GBP-dependent fast-kinetics inflammasome activation can drive the preferential processing of constitutively expressed IL-18 in IFN-γ-primed macrophages in the absence of prior Toll-like receptor stimulation. Together, our results reveal that GBPs control the kinetics of inflammasome activation and thereby shape macrophage responses toChlamydiainfections.

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Mutational Analysis of the Chlamydia muridarum Plasticity Zone

Infection and Immunity ◽

10.1128/iai.00106-15 ◽

2015 ◽

Vol 83 (7) ◽

pp. 2870-2881 ◽

Cited By ~ 29

Author(s):

Krithika Rajaram ◽

Amanda M. Giebel ◽

Evelyn Toh ◽

Shuai Hu ◽

Jasmine H. Newman ◽

...

Keyword(s):

Cell Culture ◽

Genital Tract ◽

Mutational Analysis ◽

Genomic Diversity ◽

Infection Model ◽

Plasticity Zone ◽

Content Type ◽

Chlamydia Muridarum ◽

Ifn Γ ◽

Tract Infections

Pathogenically diverseChlamydiaspp. can have surprisingly similar genomes.Chlamydia trachomatisisolates that cause trachoma, sexually transmitted genital tract infections (chlamydia), and invasive lymphogranuloma venereum (LGV) and the murine strainChlamydia muridarumshare 99% of their gene content. A region of high genomic diversity betweenChlamydiaspp. termed the plasticity zone (PZ) may encode niche-specific virulence determinants that dictate pathogenic diversity. We hypothesized that PZ genes might mediate the greater virulence and gamma interferon (IFN-γ) resistance ofC. muridarumcompared toC. trachomatisin the murine genital tract. To test this hypothesis, we isolated and characterized a series ofC. muridarumPZ nonsense mutants. Strains with nonsense mutations in chlamydial cytotoxins,guaBA-add, and a phospholipase D homolog developed normally in cell culture. Two of the cytotoxin mutants were less cytotoxic than the wild type, suggesting that the cytotoxins may be functional. However, none of the PZ nonsense mutants exhibited increased IFN-γ sensitivity in cell culture or were profoundly attenuated in a murine genital tract infection model. Our results suggest thatC. muridarumPZ genes are transcribed—and some may produce functional proteins—but are dispensable for infection of the murine genital tract.

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Early Induction of Interleukin-10 Limits Antigen-Specific CD4+T Cell Expansion, Function, and Secondary Recall Responses during Persistent Phagosomal Infection

Infection and Immunity ◽

10.1128/iai.02101-14 ◽

2014 ◽

Vol 82 (10) ◽

pp. 4092-4103 ◽

Cited By ~ 5

Author(s):

Abinav Kumar Singh ◽

Nagaraja R. Thirumalapura

Keyword(s):

T Cells ◽

T Cell ◽

Functional Status ◽

Persistent Infection ◽

Inflammatory Responses ◽

Critical Role ◽

Interleukin 10 ◽

Host Cells ◽

Content Type ◽

Ifn Γ

ABSTRACTDiverse pathogens have evolved to survive and replicate in the endosomes or phagosomes of the host cells and establish persistent infection. Ehrlichiae are Gram-negative, intracellular bacteria that are transmitted by ticks. Ehrlichiae reside in the endosomes of the host phagocytic or endothelial cells and establish persistent infection in their vertebrate reservoir hosts. CD4+T cells play a critical role in protection against phagosomal infections. In the present study, we investigated the expansion, maintenance, and functional status of antigen-specific CD4+T cells during persistentEhrlichia murisinfection in wild-type and interleukin-10 (IL-10)-deficient mice. Our study indicated that early induction of IL-10 led to reduced inflammatory responses and impaired bacterial clearance during persistentEhrlichiainfection. Notably, we demonstrated that the functional production of gamma interferon (IFN-γ) by antigen-specific CD4+T cells maintained during a persistent phagosomal infection progressively deteriorates. The functional loss of IFN-γ production by antigen-specific CD4+T cells was reversed in the absence of IL-10. Furthermore, we demonstrated that transient blockade of IL-10 receptor during the T cell priming phase early in infection was sufficient to enhance the magnitude and the functional capacity of antigen-specific effector and memory CD4+T cells, which translated into an enhanced recall response. Our findings provide new insights into the functional status of antigen-specific CD4+T cells maintained during persistent phagosomal infection. The study supports the concept that a better understanding of the factors that influence the priming and differentiation of CD4+T cells may provide a basis to induce a protective immune response against persistent infections.

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Distinct Roles of Chromosome- versus Plasmid-Encoded Genital Tract Virulence Factors in PromotingChlamydia muridarumColonization in the Gastrointestinal Tract

Infection and Immunity ◽

10.1128/iai.00265-19 ◽

2019 ◽

Vol 87 (8) ◽

Cited By ~ 7

Author(s):

John J. Koprivsek ◽

Tianyuan Zhang ◽

Qi Tian ◽

Ying He ◽

Hong Xu ◽

...

Keyword(s):

Gastrointestinal Tract ◽

Virulence Factors ◽

Genital Tract ◽

Oral Delivery ◽

Content Type ◽

Chlamydia Muridarum ◽

Ifn Γ ◽

Plasmid Mutant ◽

Increased Susceptibility ◽

Do So

ABSTRACTThe genital pathogenChlamydiais known to colonize the gastrointestinal tract. Orally deliveredChlamydia muridarumcan reach the colon and maintain a long-lasting colonization there. However,C. muridarumwith mutations in chromosomal genestc0237andtc0668(designated a chromosomal mutant) or deficient in plasmid-encoded pGP3 (designated a plasmid mutant) is unable to do so. We now report that the chromosomal mutant is still able to reach the colon while the plasmid mutant fails to do so following an oral delivery, suggesting that lack of colon colonization by different mutants may involve distinct mechanisms. Consistently, a direct intracolonic delivery selectively restored the ability of the plasmid mutant, but not the chromosomal mutant, to colonize the colon. The chromosomal mutant was rescued only in the colon of mice deficient in gamma interferon (IFN-γ). Thus, the chromosomal mutant’s deficiency in colonizing colonic mucosal tissue is likely due to its increased susceptibility to IFN-γ-mediated immunity. Furthermore, IFN-γ deficiency was sufficient for rescuing colon colonization of an orally delivered chromosomal mutant but not plasmid mutant while mice deficient in gastric acid production rescued the plasmid mutant but not the chromosomal mutant. Both mutants are attenuated in inducing genital tract pathology. Thus, we propose that chlamydial chromosomal-gene-encoded genital tract virulence factors may be essential forChlamydiato maintain long-lasting colonization in the colon while the plasmid may enableChlamydiato reach the colon by promoting evasion of gastric barriers.

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Periodontal Pathogens Invade Gingiva and Aortic Adventitia and Elicit Inflammasome Activation in αvβ6 Integrin-Deficient Mice

Infection and Immunity ◽

10.1128/iai.01077-15 ◽

2015 ◽

Vol 83 (12) ◽

pp. 4582-4593 ◽

Cited By ~ 27

Author(s):

Irina M. Velsko ◽

Sasanka S. Chukkapalli ◽

Mercedes F. Rivera-Kweh ◽

Donghang Zheng ◽

Ikramuddin Aukhil ◽

...

Keyword(s):

Atherosclerotic Plaque ◽

Inflammatory Responses ◽

Periodontal Diseases ◽

Periodontal Pocket ◽

Bacterial Invasion ◽

Inflammasome Activation ◽

Periodontal Pathogens ◽

Content Type ◽

Amyloid A ◽

Significant Difference

The American Heart Association supports an association between periodontal diseases and atherosclerosis but not a causal association. This study explores the use of the integrin β6−/−mouse model to study the causality. We investigated the ability of a polymicrobial consortium ofPorphyromonas gingivalis,Treponema denticola,Tannerella forsythia, andFusobacterium nucleatumto colonize the periodontium and induce local and systemic inflammatory responses. Polymicrobially infectedItgβ6−/−mice demonstrate greater susceptibility to gingival colonization/infection, with severe gingival inflammation, apical migration of the junctional epithelium, periodontal pocket formation, alveolar bone resorption, osteoclast activation, bacterial invasion of the gingiva, a greater propensity for the bacteria to disseminate hematogenously, and a strong splenic T cell cytokine response. Levels of atherosclerosis risk factors, including serum nitric oxide, oxidized low-density lipoprotein, serum amyloid A, and lipid peroxidation, were significantly altered by polybacterial infection, demonstrating an enhanced potential for atherosclerotic plaque progression. Aortic gene expression revealed significant alterations in specific Toll-like receptor (TLR) and nucleotide-binding domain- and leucine-rich-repeat-containing receptor (NLR) pathway genes in response to periodontal bacterial infection. Histomorphometry of the aorta demonstrated larger atherosclerotic plaques inItgβ6−/−mice than in wild-type (WT) mice but no significant difference in atherosclerotic plaque size between mice with polybacterial infection and mice with sham infection. Fluorescencein situhybridization demonstrated active invasion of the aortic adventitial layer byP. gingivalis. Our observations suggest that polybacterial infection elicits distinct aortic TLR and inflammasome signaling and significantly increases local aortic oxidative stress. These results are the first to demonstrate the mechanism of the host aortic inflammatory response induced by polymicrobial infection with well-characterized periodontal pathogens.

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Neutrophils Mediate Immunopathology and Negatively Regulate Protective Immune Responses during Fatal Bacterial Infection-Induced Toxic Shock

Infection and Immunity ◽

10.1128/iai.01409-12 ◽

2013 ◽

Vol 81 (5) ◽

pp. 1751-1763 ◽

Cited By ~ 29

Author(s):

Qin Yang ◽

Purnima Ghose ◽

Nahed Ismail

Keyword(s):

Bacterial Infection ◽

Inflammatory Responses ◽

Nkt Cells ◽

Intracellular Bacterium ◽

Transcriptional Analysis ◽

Toxic Shock ◽

Necrosis Factor Alpha ◽

Content Type ◽

Neutrophil Depletion ◽

Ifn Γ

ABSTRACTEhrlichia chaffeensisis an obligate intracellular bacterium that infects primarily monocytes and macrophages and causes potentially fatal human monocytic ehrlichiosis (HME) that mimics toxic-shock-like syndrome in immunocompetent hosts. Early recruitment of neutrophils to the sites of infection is critical for the control of bacterial infection and inflammatory responses. We recently observed rapid and sustained neutrophil recruitment at a primary site of infection (peritoneum) following lethal murine ehrlichial infection compared to innocuous ehrlichial infection. We examined here the contribution of neutrophils to protective immunity or immunopathology during infection with monocyticEhrlichia. Unexpectedly, depletion of neutrophils from lethally infected mice enhanced bacterial elimination, decreased immune-mediated pathology, and prolonged survival. Furthermore, compared to lethally infected sham controls, neutrophil depletion in infected mice resulted in amelioration of pathogenic responses, as evidenced by a decreased number of tumor necrosis factor alpha (TNF-α)-producing CD8+T cells, which is known to mediate immunopathology and toxic shock in a murine model of fatal ehrlichiosis. Although neutrophil depletion did not influence the number of CD4+Th1 cells and NKT cells producing gamma interferon (IFN-γ), it increased the ratio of IFN-γ- to IL-10-producing NKT cells as well as the ratio of IFN-γ to interleukin 10 (IL-10) transcripts in the liver. This may ameliorate the net suppressive effect of IL-10 on IFN-γ-mediated activation of infected macrophages and thus may account for the enhanced bacterial elimination. Finally, transcriptional analysis of gene expression in the liver indicated that neutrophils contribute to overproduction of cytokines and chemokines during fatal ehrlichiosis. In conclusion, these results revealed an unexpected role of neutrophils in supporting bacterial replication indirectly and promoting immunopathology during severe infection with an intracellular bacterium.

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Human GBP1 promotes pathogen vacuole rupture and inflammasome activation during Legionella pneumophila infection

10.1101/2020.05.27.120477 ◽

2020 ◽

Author(s):

Antonia R. Bass ◽

Sunny Shin

Keyword(s):

Legionella Pneumophila ◽

Severe Pneumonia ◽

Inflammasome Activation ◽

Dependent Manner ◽

Cytokine Release ◽

Legionnaires Disease ◽

Cell Cytosol ◽

Ifn Γ ◽

Host Cell Cytosol ◽

Family Cytokine

AbstractThe inflammasome is an essential component of host defense against intracellular bacterial pathogens, such as Legionella pneumophila, the causative agent of the severe pneumonia Legionnaires’ disease. Inflammasome activation leads to recruitment and activation of caspases, which promote IL-1 family cytokine release and pyroptosis. In mice, interferon (IFN) signaling promotes inflammasome responses against L. pneumophila, in part through the functions of a family of IFN-inducible GTPases known as guanylate binding proteins (GBPs) (1). Within murine macrophages, IFN signaling promotes rupture of the L. pneumophila-containing vacuole (LCV), whereas GBPs are dispensable for vacuole rupture. Instead, GBPs facilitate the lysis of cytosol-exposed L. pneumophila. In contrast to mouse GBPs, the functions of human GBPs in inflammasome responses to L. pneumophila are poorly understood. Here, we show that IFN-γ promotes caspase-1, caspase-4, and caspase-5 inflammasome activation during L. pneumophila infection and upregulates GBP expression in primary human macrophages. We find that human GBP1 is important for maximal IFN-γ-driven inflammasome responses to L. pneumophila. Furthermore, IFN-γ signaling promotes the rupture of LCVs. Intriguingly, in contrast to murine GBPs, human GBP1 targets the LCV in a T4SS-dependent manner and promotes vacuolar lysis, resulting in increased bacterial access to the host cell cytosol. Our findings show a key role for human GBP1 in targeting and disrupting pathogen-containing vacuoles and reveal mechanistic differences in how mouse and human GBPs promote inflammasome responses to L. pneumophila.

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IRGM3 Contributes to Immunopathology and Is Required for Differentiation of Antigen-Specific Effector CD8+T Cells in Experimental Cerebral Malaria

Infection and Immunity ◽

10.1128/iai.02701-14 ◽

2015 ◽

Vol 83 (4) ◽

pp. 1406-1417 ◽

Cited By ~ 5

Author(s):

Jintao Guo ◽

James A. McQuillan ◽

Belinda Yau ◽

Gregory S. Tullo ◽

Carole A. Long ◽

...

Keyword(s):

T Cells ◽

Cerebral Malaria ◽

Inflammatory Cytokines ◽

Inflammatory Responses ◽

Type I ◽

Experimental Cerebral Malaria ◽

Content Type ◽

Cytokines And Chemokines ◽

Specific Effector ◽

Ifn Γ

Gamma interferon (IFN-γ) drives antiparasite responses and immunopathology during infection withPlasmodiumspecies. Immunity-related GTPases (IRGs) are a class of IFN-γ-dependent proteins that are essential for cell autonomous immunity to numerous intracellular pathogens. However, it is currently unknown whether IRGs modulate responses during malaria. We have used thePlasmodium bergheiANKA (PbA) model in which mice develop experimental cerebral malaria (ECM) to study the roles of IRGM1 and IRGM3 in immunopathology. Induction of mRNA forIrgm1andIrgm3was found in the brains and spleens of infected mice at times of peak IFN-γ production.Irgm3−/−but notIrgm1−/−mice were completely protected from the development of ECM, and this protection was associated with the decreased induction of inflammatory cytokines, as well as decreased recruitment and activation of CD8+T cells within the brain. Although antigen-specific proliferation of transferred CD8+T cells was not diminished compared to that of wild-type recipients following PbA infection, T cells transferred intoIrgm3−/−recipients showed a striking impairment of effector differentiation. Decreased induction of several inflammatory cytokines and chemokines (interleukin-6, CCL2, CCL3, and CCL4), as well as enhanced mRNA expression of type-I IFNs, was found in the spleens ofIrgm3−/−mice at day 4 postinfection. Together, these data suggest that protection from ECM pathology inIrgm3−/−mice occurs due to impaired generation of CD8+effector function. This defect is nonintrinsic to CD8+T cells. Instead, diminished T cell responses most likely result from defective initiation of inflammatory responses in myeloid cells.

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TLR11-independent inflammasome activation is critical for CD4+ T cell-derived IFN-γ production and host resistance to Toxoplasma gondii

PLoS Pathogens ◽

10.1371/journal.ppat.1007872 ◽

2019 ◽

Vol 15 (6) ◽

pp. e1007872 ◽

Cited By ~ 11

Author(s):

Américo H. López-Yglesias ◽

Ellie Camanzo ◽

Andrew T. Martin ◽

Alessandra M. Araujo ◽

Felix Yarovinsky

Keyword(s):

T Cell ◽

Toxoplasma Gondii ◽

Host Resistance ◽

Cd4 T Cell ◽

Inflammasome Activation ◽

Ifn Γ

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Guanylate Binding Proteins Regulate Inflammasome Activation in Response to Hyperinjected Yersinia Translocon Components

Infection and Immunity ◽

10.1128/iai.00778-16 ◽

2017 ◽

Vol 85 (10) ◽

Cited By ~ 22

Author(s):

Erin E. Zwack ◽

Eric M. Feeley ◽

Amanda R. Burton ◽

Baofeng Hu ◽

Masahiro Yamamoto ◽

...

Keyword(s):

Binding Proteins ◽

Membrane Damage ◽

Effector Proteins ◽

Host Cells ◽

Target Cells ◽

Inflammasome Activation ◽

Secretion Systems ◽

Content Type ◽

Endosomal Membrane ◽

Galectin 3

ABSTRACT Gram-negative bacterial pathogens utilize virulence-associated secretion systems to inject, or translocate, effector proteins into host cells to manipulate cellular processes and promote bacterial replication. However, translocated bacterial products are sensed by nucleotide binding domain and leucine-rich repeat-containing proteins (NLRs), which trigger the formation of a multiprotein complex called the inflammasome, leading to secretion of interleukin-1 (IL-1) family cytokines, pyroptosis, and control of pathogen replication. Pathogenic Yersinia bacteria inject effector proteins termed Yops, as well as pore-forming proteins that comprise the translocon itself, into target cells. The Yersinia translocation regulatory protein YopK promotes bacterial virulence by limiting hyperinjection of the translocon proteins YopD and YopB into cells, thereby limiting cellular detection of Yersinia virulence activity. How hyperinjection of translocon proteins leads to inflammasome activation is currently unknown. We found that translocated YopB and YopD colocalized with the late endosomal/lysosomal protein LAMP1 and that the frequency of YopD and LAMP1 association correlated with the level of caspase-1 activation in individual cells. We also observed colocalization between YopD and Galectin-3, an indicator of endosomal membrane damage. Intriguingly, YopK limited the colocalization of Galectin-3 with YopD, suggesting that YopK limits the induction or sensing of endosomal membrane damage by components of the type III secretion system (T3SS) translocon. Furthermore, guanylate binding proteins (GBPs) encoded on chromosome 3 (Gbp Chr3 ), which respond to pathogen-induced damage or alteration of host membranes, were necessary for inflammasome activation in response to hyperinjected YopB/-D. Our findings indicate that lysosomal damage by Yersinia translocon proteins promotes inflammasome activation and implicate GBPs as key regulators of this process.

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Vaccine-Specific Immune Responses against Mycobacterium ulcerans Infection in a Low-Dose Murine Challenge Model

Infection and Immunity ◽

10.1128/iai.00753-19 ◽

2019 ◽

Vol 88 (3) ◽

Cited By ~ 1

Author(s):

Kirstie M. Mangas ◽

Andrew H. Buultjens ◽

Jessica L. Porter ◽

Sarah L. Baines ◽

Estelle Marion ◽

...

Keyword(s):

Low Dose ◽

Inflammatory Responses ◽

Subcutaneous Tissue ◽

Interleukin 2 ◽

Mycobacterium Ulcerans ◽

Prognostic Models ◽

Effective Vaccine ◽

Infection Model ◽

Content Type ◽

Ifn Γ

ABSTRACT The neglected tropical disease Buruli ulcer (BU) is an infection of subcutaneous tissue with Mycobacterium ulcerans. There is no effective vaccine. Here, we assessed an experimental prime-boost vaccine in a low-dose murine tail infection model. We used the enoyl reductase (ER) domain of the M. ulcerans mycolactone polyketide synthases electrostatically coupled with a previously described Toll-like receptor 2 (TLR-2) agonist-based lipopeptide adjuvant, R4Pam2Cys. Mice were vaccinated and then challenged via tail inoculation with 14 to 20 CFU of a bioluminescent strain of M. ulcerans. Mice receiving either the experimental ER vaccine or Mycobacterium bovis bacillus Calmette-Guérin (BCG) were equally protected, with both groups faring significantly better than nonvaccinated animals (P < 0.05). To explore potential correlates of protection, a suite of 29 immune parameters were assessed in the mice at the end of the experimental period. Multivariate statistical approaches were used to interrogate the immune response data to develop disease-prognostic models. High levels of interleukin 2 (IL-2) and low gamma interferon (IFN-γ) produced in the spleen best predicted control of infection across all vaccine groups. Univariate logistic regression revealed vaccine-specific profiles of protection. High titers of ER-specific IgG serum antibodies together with IL-2 and IL-4 in the draining lymph node (DLN) were associated with protection induced by the ER vaccine. In contrast, high titers of IL-6, tumor necrosis factor alpha (TNF-α), IFN-γ, and IL-10 in the DLN and low IFN-γ titers in the spleen were associated with protection following BCG vaccination. This study suggests that an effective BU vaccine must induce localized, tissue-specific immune profiles with controlled inflammatory responses at the site of infection.

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