Inflammasomes are cytosolic protein complexes, which orchestrate the maturation of active IL‐1β by proteolytic cleavage via caspase‐1. Although many principles of inflammasome activation have been ...described, mechanisms that limit inflammasome‐dependent immune responses remain poorly defined. Here, we show that the thiol‐specific peroxidase peroxiredoxin‐4 (Prdx4) directly regulates IL‐1β generation by interfering with caspase‐1 activity. We demonstrate that caspase‐1 and Prdx4 form a redox‐sensitive regulatory complex via caspase‐1 cysteine 397 that leads to caspase‐1 sequestration and inactivation. Mice lacking Prdx4 show an increased susceptibility to LPS‐induced septic shock. This effect was phenocopied in mice carrying a conditional deletion of Prdx4 in the myeloid lineage (Prdx4‐ΔLysMCre). Strikingly, we demonstrate that Prdx4 co‐localizes with inflammasome components in extracellular vesicles (EVs) from inflammasome‐activated macrophages. Purified EVs are able to transmit a robust IL‐1β‐dependent inflammatory response in vitro and also in recipient mice in vivo. Loss of Prdx4 boosts the pro‐inflammatory potential of EVs. These findings identify Prdx4 as a critical regulator of inflammasome activity and provide new insights into remote cell‐to‐cell communication function of inflammasomes via macrophage‐derived EVs.
Synopsis
This study shows that the thiol‐specific peroxidase peroxiredoxin‐4 (Prdx4) directly regulates IL‐1β generation by interfering with inflammasome activity. Prdx4 forms a redox‐sensitive regulatory complex with the caspase‐1 at the cysteine in position 397 that leads to caspase‐1 sequestration and inactivation.
Lack of Prdx4 in myeloid cells leads to elevated IL‐1β levels and increased clinical symptoms in a murine septic shock model.
Prdx4 interacts with caspase‐1 in the cytosolic compartment and in extracellular vesicles (EVs) of activated macrophages.
Purified EVs from macrophages contain all components of the NLRP3 inflammasome as well as Prdx4 and are able to convey a robust IL‐1β‐dependent inflammatory response in vitro and in vivo.
Loss of Prdx4 leads to an increased pro‐inflammatory potential of EVs, indicating a potential role of Prdx4 in the regulation of cell‐to‐cell communication via macrophage‐derived EVs.
The thiol‐specific peroxidase Prdx4 directly regulates caspase‐1 activity in a redox‐sensitive manner to control IL‐1β maturation.
Purpose
Transport of secretory immunoglobulin A (SIgA) through the airway epithelial cell barrier into the mucosal lumen by the polymeric immunoglobulin receptor (pIgR) is an important mechanism of ...respiratory mucosal host defense. Identification of immunomodulating substances that regulate secretory immunity might have therapeutic implications with regard to an improved immune exclusion.
Thus, we sought to analyze secretory immunity under homeostatic and immunomodulating conditions in different compartments of the murine upper and lower respiratory tract (URT&LRT).
Methods
Pigr
gene expression in lung, trachea, and nasal-associated lymphoid tissue (NALT) of germ-free mice, specific pathogen-free mice, mice with an undefined microbiome, as well as LPS- and IFN-γ-treated mice was determined by quantitative real-time PCR. IgA levels in bronchoalveolar lavage (BAL), nasal lavage (NAL), and serum were determined by ELISA. LPS- and IFN-γ-treated mice were colonized with
Streptococcus pneumoniae
and bacterial CFUs were determined in URT and LRT.
Results
Respiratory
Pigr
expression and IgA levels were dependent on the degree of exposure to environmental microbial stimuli. While immunostimulation with LPS and IFN-γ differentially impacts respiratory
Pigr
expression and IgA in URT
vs
. LRT, only prophylactic IFN-γ treatment reduces nasal colonization with
S. pneumoniae
.
Conclusion
Airway-associated secretory immunity can be partly modulated by exposure to microbial ligands and proinflammatory stimuli. Prophylactic IFN-γ-treatment modestly improves antibacterial immunity in the URT, but this does not appear to be mediated by SIgA or pIgR.
MyD88-mediated signaling downstream of Toll-like receptors and the IL-1 receptor family is critically involved in the induction of protective host responses upon infections. Although it is known that ...MyD88-deficient mice are highly susceptible to a wide range of bacterial infections, the cell type-specific contribution of MyD88 in protecting the host against intestinal bacterial infection is only poorly understood. In order to investigate the importance of MyD88 in specific immune and nonimmune cell types during intestinal infection, we employed a novel murine knock-in model for MyD88 that enables the cell type-specific reactivation of functional MyD88 expression in otherwise MyD88-deficient mice. We report here that functional MyD88 signaling in CD11c+ cells was sufficient to activate intestinal dendritic cells (DC) and to induce the early group 3 innate lymphoid cell (ILC3) response as well as the development of colonic Th17/Th1 cells in response to infection with the intestinal pathogen C. rodentium. In contrast, restricting MyD88 signaling to several other cell types, including macrophages (MO), T cells or ILC3 did not induce efficient intestinal immune responses upon infection. However, we observed that the functional expression of MyD88 in intestinal epithelial cells (IEC) also partially protected the mice during intestinal infection, which was associated with enhanced epithelial barrier integrity and increased expression of the antimicrobial peptide RegIIIγ and the acute phase protein SAA1 by epithelial cells. Together, our data suggest that MyD88 signaling in DC and IEC is both essential and sufficient to induce a full spectrum of host responses upon intestinal infection with C. rodentium.
Human hemato-lymphoid-system mice hold great promise for modeling and studying important human diseases in vivo, and to enable vaccine testing. Until now, several major limitations have restricted ...the utility of human hemato-lymphoid-system mice in translational research. Recently, however, significant advances have been made in improving these mice, based on the delivery of human cytokines to create a better environment for human cells in the mouse host. In this review, we discuss the various approaches with a particular focus on improving human hemato-lymphoid-system mice by human cytokine knock-in gene replacement.
Dendritic cells (DCs) are known to induce the growth and function of natural killer (NK) cells. Here, we address the capacity of DCs to interact with NK cells in human lymphoid organs and identify ...the role of specific DC-derived cytokines. We demonstrate that DCs colocalize with NK cells in the T cell areas of lymph nodes. In culture, DCs from either blood or spleen primarily stimulate the CD56brightCD16-NK cell subset, which is enriched in secondary lymphoid tissues. Blocking of IL-12 abolished DC-induced IFN-γ secretion by NK cells, whereas membrane-bound IL-15 on DCs was essential for NK cell proliferation and survival. Maturation by CD40 ligation promoted the highest IL-15 surface presentation on DCs and led to the strongest NK cell proliferation induced by DCs. These results identify secondary lymphoid organs as a potential DC/NK cell interaction site and identify the distinct roles for DC-derived IL-12 and IL-15 in NK cell activation.
In recent years, the gut–central nervous system axis has emerged as a key factor in the pathophysiology of spinal cord injury (SCI). Interleukin-13 (IL-13) has been shown to have anti-inflammatory ...and neuroprotective effects in SCI. The aim of this study was to investigate the changes in microbiota composition after hemisection injury and to determine whether systemic recombinant (r)IL-13 treatment could alter the gut microbiome, indirectly promoting functional recovery. The gut microbiota composition was determined by 16S rRNA gene sequencing, and correlations between gut microbiota alterations and functional recovery were assessed. Our results showed that there were no changes in alpha diversity between the groups before and after SCI, while PERMANOVA analysis for beta diversity showed significant differences in fecal microbial communities. Phylogenetic classification of bacterial families revealed a lower abundance of the Bacteroidales S24-7 group and a higher abundance of Lachnospiraceae and Lactobacillaceae in the post-SCI group. Systemic rIL-13 treatment improved functional recovery 28 days post-injury and microbiota analysis revealed increased relative abundance of Clostridiales vadin BB60 and Acetitomaculum and decreased Anaeroplasma, Ruminiclostridium_6, and Ruminococcus compared to controls. Functional assessment with PICRUSt showed that genes related to glyoxylate cycle and palmitoleate biosynthesis-I were the predominant signatures in the rIL-13-treated group, whereas sulfolactate degradation super pathway and formaldehyde assimilation-I were enriched in controls. In conclusion, our results indicate that rIL-13 treatment promotes changes in gut microbial communities and may thereby contribute indirectly to the improvement of functional recovery in mice, possibly having important implications for the development of novel treatment options for SCI.
Many bacteria move using a complex, self-assembling nanomachine, the bacterial flagellum. Biosynthesis of the flagellum depends on a flagellar-specific type III secretion system (T3SS), a protein ...export machine homologous to the export machinery of the virulence-associated injectisome. Six cytoplasmic (FliH/I/J/G/M/N) and seven integral-membrane proteins (FlhA/B FliF/O/P/Q/R) form the flagellar basal body and are involved in the transport of flagellar building blocks across the inner membrane in a proton motive force-dependent manner. However, how the large, multi-component transmembrane export gate complex assembles in a coordinated manner remains enigmatic. Specific for most flagellar T3SSs is the presence of FliO, a small bitopic membrane protein with a large cytoplasmic domain. The function of FliO is unknown, but homologs of FliO are found in >80% of all flagellated bacteria. Here, we demonstrate that FliO protects FliP from proteolytic degradation and promotes the formation of a stable FliP-FliR complex required for the assembly of a functional core export apparatus. We further reveal the subcellular localization of FliO by super-resolution microscopy and show that FliO is not part of the assembled flagellar basal body. In summary, our results suggest that FliO functions as a novel, flagellar T3SS-specific chaperone, which facilitates quality control and productive assembly of the core T3SS export machinery.
Colibactin, a bacterial genotoxin produced by E. coli strains harboring the pks genomic island, induces cytopathic effects, such as DNA breaks, cell cycle arrest, and apoptosis. Patients with ...inflammatory bowel diseases, such as ulcerative colitis, display changes in their microbiota with the expansion of E. coli. Whether and how colibactin affects the integrity of the colonic mucosa and whether pks+ E. coli contributes to the pathogenesis of colitis is not clear. Using a gnotobiotic mouse model, we show that under homeostatic conditions, pks+ E. coli do not directly interact with the epithelium or affect colonic integrity. However, upon short-term chemical disruption of mucosal integrity, pks+ E. coli gain direct access to the epithelium, causing epithelial injury and chronic colitis, while mice colonized with an isogenic ΔclbR mutant incapable of producing colibactin show a rapid recovery. pks+ E. coli colonized mice are unable to reestablish a functional barrier. In turn, pks+ E. coli remains in direct contact with the epithelium, perpetuating the process and triggering chronic mucosal inflammation that morphologically and transcriptionally resembles human ulcerative colitis. This state is characterized by impaired epithelial differentiation and high proliferative activity, which is associated with high levels of stromal R-spondin 3. Genetic overexpression of R-spondin 3 in colon myofibroblasts is sufficient to mimic barrier disruption and expansion of E. coli. Together, our data reveal that pks+ E. coli are pathobionts that promote severe injury and initiate a proinflammatory trajectory upon contact with the colonic epithelium, resulting in a chronic impairment of tissue integrity.
Campylobacter jejuni
is the most common bacterial cause of foodborne zoonosis in the European Union. Infections are often linked to the consumption and handling of poultry meat. The aim of the ...present study was to investigate the caecal microbiota of birds infected with
C. jejuni
at different ages. Therefore, a total of 180 birds of the laying hybrid Lohmann Brown-Classic were housed in 12 subgroups of 15 animals each in three performed repetitions. Three birds per subgroup were experimentally infected with
C. jejuni
at an age of about 21 days and about 78 days (4.46 ± 0.35 log
10
CFU/bird). Twenty-one days after experimental infection, microbiome studies were performed on 72 caecal samples of dissected birds (three primary infected and three further birds/subgroup). Amplification within the hypervariable region V 4 of the 16S rRNA gene was performed and sequenced with the Illumina MiSeq platform. Statistical analyses were performed using SAS
®
Enterprise Guide
®
(version 7.1) and R (version 3.5.2). Both factors, the experimental replication (
p
< 0.001) and the chickens’ age at infection (
p
< 0.001) contributed significantly to the differences in microbial composition of the caecal samples. The factor experimental replication explained 24% of the sample’s variability, whereas the factor age at infection explained 14% thereof. Twelve of 32 families showed a significantly different count profile between the two age groups, whereby strongest differences were seen for seven families, among them the family
Campylobacteraceae
(adjusted
p
= 0.003). The strongest difference between age groups was seen for a bacterial species that is assigned to the genus
Turicibacter
which in turn belongs to the family
Erysipelotrichaceae
(adjusted
p
< 0.0001). Correlation analyses revealed a common relationship in both chicken ages at infection between the absolute abundance of
Campylobacteraceae
and
Alcaligenaceae
, which consists of the genus
Parasutterella.
In general, concentrations of particular volatile fatty acids (VFA) demonstrated a negative correlation to absolute abundance of
Campylobacteraceae
, whereby the strongest link was seen for n-butyrate (−0.51141;
p
< 0.0001). Despite performing consecutive repetitions, the factor experimental replication contributed more to the differences of microbial composition in comparison to the factor age at infection.
In vivo models that recapitulate human erythropoiesis with persistence of circulating red blood cells (RBCs) have remained elusive. We report an immunodeficient murine model in which combined human ...liver and cytokine humanization confer enhanced human erythropoiesis and RBC survival in the circulation. We deleted the fumarylacetoacetate hydrolase (
) gene in MISTRG mice expressing several human cytokines in place of their murine counterparts. Liver humanization by intrasplenic injection of human hepatocytes (huHep) eliminated murine complement C3 and reduced murine Kupffer cell density. Engraftment of human sickle cell disease (SCD)-derived hematopoietic stem cells in huHepMISTRG
mice resulted in vaso-occlusion that replicated acute SCD pathology. Combined liver-cytokine-humanized mice will facilitate the study of diseases afflicting RBCs, including bone marrow failure, hemoglobinopathies, and malaria, and also preclinical testing of therapies.
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