Abstract
Despite recent advances in understanding the role of IL-17 in host immunity, its role in regulating enteric immune responses as well as its impact on the commensal microbiome has not been ...well studied. To further understand this, we have generated intestinal epithelial cell specific IL-17R knockout mice.
Our data shows that global and intestinal epithelial cell specific Il17r−/− mice have overgrowth of segmented filamentous bacteria (SFB), suggesting a critical role of IL-17 signaling in SFB colonization. Higher SFB colonization in Il17rafl/fl x villin cre+ mice resulted in expansion of IL-17A and IL-22 producing Th17 cells. The expansion of SFB was also confirmed by 16S rRNA. Furthermore this analysis also showed higher abundance of S24-7 and the Clostridiales family in Il17rafl/fl x villin cre+ mice. RNA sequencing data from the distal small intestine of SFB-colonized Il17ra−/− and Il17rafl/fl x villin cre+ mice revealed substantial reduction of Nox1 (an apical NADPH oxidase) and Pigr genes. Mouse and human primary intestinal organoid culture further confirmed a direct role of IL-17 in regulating Nox1 and Pigr expression. Reduced Nox1 expression correlated with significant reduction in H202 levels in the terminal ileum of Il17rcfl/fl x villin cre+ mice. Furthermore, Nox1−/− and IgA−/− mice showed a higher degree of SFB colonization in the feces and terminal ileum as compared to cohoused control WT mice. We have evidence that Il17rafl/fl x villin cre+ mice are more susceptible to autoimmune inflammation.
Collectively, our data indicate IL-17R-dependent intestinal signaling controls commensal bacteria by regulating the expression of Nox1 and Pigr which regulates lumenal H202 and sIgA concentrations respectively.
Abstract
Bacterial pneumonia is a leading cause of mortality and one major pathogen associated with this disease is Klebsiella pneumoniae (KP). Thus, a vaccine against KP is in great need especially ...for immunocompromised patients. Here we show that intranasal immunization of mice with heat-killed KP strongly induces IL-17 production in the lung. Using an Il17f reporter mouse, we were able to sort IL-17F positive cells after immunization by FACS. Further analysis reveals that these cells are conventional CD4+TCR-beta+ Th17 cells and they express Il17a, Il17f, Il22, Rora and Rorc. These Th17 cells are antigen specific as they proliferate and produce IL-17 upon re-stimulation with heat-killed KP but not heat-killed Escherichia coli or Staphylococcus aureus. These responses ultimately lead to the protection of immunized mice from live KP infection. This immunization strategy also confers protection in B cell-deficient (mu-MT) mice, demonstrating the vaccination can be antibody independent. We also show that other mucosal routes of vaccination such as intragastric immunization also reduces bacterial burden during pulmonary infection, suggesting that Th17 cells can be primed or recruited from other mucosal sites distal to the lung and oral vaccine can be effective in pulmonary infection. These data suggest that the Th17 pathway can be a target of pulmonary vaccine development through intranasal or intragastric immunization.
Targeted, specific receptor mediated gene transfer is a major goal of gene therapy research to accomplish gene transfer exclusively to the desired cell population.
First, the use of natural receptor ...for stem cell factor and transferrin receptor-targeted gene transfer using poly-L-lysine-based molecular conjugate vectors was evaluated in a panel of hematopoietic progenitor cell lines. Second, the ability of poly-L-lysine to enhance adenovirus mediated gene transfer efficiency was examined in different cell lines by using recombinant adenovirus-poly-L-lysine molecular conjugate conglomerates (recMCVEGFP).
Despite effective ligand internalization receptor, gene expression amplification in receptor positive cell lines was not uniformly observed. Therefore, using a poly-L-lysine-based, receptor-targeted vector, neither transferrin nor natural receptor for stem cell factor mediated gene transfer can be considered a universally applicable procedure that exclusively depends on the presence of receptors on the cell surface; rather, it is a cell specific phenomenon. In our model, poly-L-lysine is the major contributor for gene transfer to hematopoietic progenitor cells, mediating the initial vector-cell binding. Human progenitor cell lines are poorly transduceable with recombinant adenovirus vectors. This new poly-L-lysine-modified, adenovirus-based vector could overcome virus tropism restrictions and consistently achieve very high transduction efficiency (>90%) in cells otherwise refractory to adenovirus gene transfer.
Polylysine-based adenovirus vectors may have promise for situations in which high-efficiency gene transfer with transient high level transgene expression in hematopoietic cells is needed, such as leukemia vaccine protocols or for purging strategies in leukemia cell contaminated stem cell preparations.
Acute hemorrhage commonly leads to coagulopathy and organ dysfunction or failure. Recent evidence suggests that damage to the endothelial glycocalyx contributes to these adverse outcomes. The ...physiological events mediating acute glycocalyx shedding are undefined, however. Here, we show that succinate accumulation within endothelial cells drives glycocalyx degradation through a membrane reorganization-mediated mechanism. We investigated this mechanism in a cultured endothelial cell hypoxia-reoxygenation model, in a rat model of hemorrhage, and in trauma patient plasma samples. We found that succinate metabolism by succinate dehydrogenase mediates glycocalyx damage through lipid oxidation and phospholipase A2-mediated membrane reorganization, promoting the interaction of matrix metalloproteinase 24 (MMP24) and MMP25 with glycocalyx constituents. In a rat hemorrhage model, inhibiting succinate metabolism or membrane reorganization prevented glycocalyx damage and coagulopathy. In patients with trauma, succinate levels were associated with glycocalyx damage and the development of coagulopathy, and the interaction of MMP24 and syndecan-1 was elevated compared to healthy controls.
Klebsiella (K.)pneumoniae is an important cause of Gram-negative nosocomial infections. Recent worldwide emergence of K. pneumoniae producing carbapenemase (KPC) poses a significant problem for stem ...cell transplantation (SCT) recipients and patients whose immunity is impaired. Such patients have few treatment options and face mortality rates over 50%. Novel strategies are needed. Emerging evidence suggests that, in addition to granulocytes, Th17 cells and IL-17 augment immunity against many bacteria including K. pneumonia. Th17 cells bridge innate and adaptive responses preventing bacterial translocation by maintaining tight intestinal mucosal junctions, promote hematopoietic stem cell function, myelopoiesis, and recruitment of myeloid cells. In an animal model, antigen-specific MHC class II-restricted Th17 cells recognizing K. pneumoniae outer membrane protein (Omp). These T cells provide serotype-independent protective mucosal immunity against K. pneumoniae, including the multi-drug resistant strains. Here we studied the endogenous reactivity of T cells of normal healthy donors against Klebsiella antigens. PBL were exposedtorecombinant K. pneumoniae outer membrane protein X (OmpX) and lysate of K. pneumoniae serotype 2 (KP). Flow cytometry revealed intracellular production of IL-17A by CD4+ T cells selectively in 4 out of 5 donors upon stimulation with KP lysate and in 3 out of 5 donors upon stimulation with OmpX protein. The observed Th17 reactivity was confined only to the effector memory compartment (TEM), suggesting an antigen-specific mechanism, but not to unrelated virus and tumor antigen control peptide libraries indicating pre-established antigen-specific immunity against K. pneumoniae in normal healthy donors.
Next the feasibility of expanding the Klebsiella-specific CD4+ T cells in vitro under Th17-polarizing conditions was tested. Magnetic bead purified CD4+ memory T cells from normal donors were co-cultured for 10-14 days with autologous monocytes or irradiated PBMCs pulsed with KP lysate, recombinant OmpX or control peptide libraries derived from BK virus. Cultures were maintained in media containing Th17 polarizing cytokines (TGF-1, IL-1β, IL-6, and IL-23) and supplemented with IL-7, IL-15, and IL-2. Two rounds of stimulation were performed and resulting T cells were tested by FACS for their ability to recognize target antigens. Both KP-lysate and OmpX-stimulated T cells from majority of the donors demonstrated robust antigen-specific production of IL-17A, TNF-α, and IFN-γ upon exposure to respective cognate antigens with some cross-reactivity between them. Control autologous BK-virus specific T cells showed minimal reactivity against KP lysate or OmpX, underscoring antigen-specific nature of observed antibacterial responses. Comparable induction of anti-KP responses was achieved using CD4+ T cells from pre-transplant patients with leukemia, but there was marked impairment when T cells isolated from recipients of allogeneic SCT were used. Importantly, T cells generated using KP serotype 2 lysate demonstrated robust recognition of KP lysate from KP396 (serotype 1), while significantly lower cross-reactivity was observed against lysate from E. coli and no responses were seen upon stimulation with antigens from Gram-positive bacteria (S. aureus and S. pneumoniae),
In summary, for the first time we demonstrate that human Th17 cells from healthy donors specifically recognize K. pneumoniae OMP, analogous to the animal model of Th17-mediated serotype-independent immunity against K. pneumoniae. Notably, we efficiently expanded the KP-reactive CD4+ Th17 cells in vitro and these cells recognized KP antigens independently of serotype, indicating the feasibility of targeting simultaneously many strains of KP including the multi-drug-resistant isolates. These results raise the possibility of testing adoptively-transferred antibacterial Th17 cells as a novel strategy to augment host defenses of patients colonized with antibiotic-resistant bacteria at risk for Gram-negative sepsis.
No relevant conflicts of interest to declare.
Pneumonia is a leading cause of death worldwide.
Staphylococcal aureus
can be a cause of severe pneumonia alone or as a common pathogen in secondary pneumonia following influenza. Recently, we have ...reported that preceding influenza attenuated the Type 17 pathway, increasing the lung’s susceptibility to secondary infection. IL-1β is known to regulate host defense including playing a role in T
H
17 polarization. We examined if IL-1β signaling is required for
S. aureus
host defense and whether influenza infection impacted
S. aureus
induced IL-1β production and subsequent Type 17 pathway activation. Mice were challenged with
S. aureus
(USA300) with or without preceding influenza A/PR/8/34 H1N1 infection. IL-1receptor1 −/− mice had significantly higher
S. aureus
burden, increased mortality, and decreased Type 17 pathway activation following
S. aureus
challenge. Co-infected mice had significantly decreased IL-1β production versus
S. aureus
alone at early time points following bacterial challenge. Preceding influenza did not attenuate
S. aureus
induced inflammasome activation, but there was early suppression of NF-κB activation, suggesting an inhibition of NF-κB dependent transcription of pro- IL-1β. Furthermore, overexpression of IL-1β in influenza,
S. aureus
co-infected mice rescued the induction of IL-17 and IL-22 by
S. aureus
and improved bacterial clearance. Finally, exogenous IL-1β did not significantly rescue
S. aureus
host defense during co-infection in IL-17RA −/− mice or in mice in which IL-17 and IL-22 activity were blocked. These data reveal a novel mechanism by which influenza A inhibits
S. aureus
induced IL-1β production resulting in attenuation of Type 17 immunity and increased susceptibility to bacterial infection.
The limitations of currently available treatment for severe respiratory infection are demonstrated by the relatively fixed mortality associated with these infections despite advances in nutrition, ...vaccines, antibiotics, and critical care. This might be due in part to the changing spectrum of pathogens and development of drug resistance. Cytokines are potent molecules that function as growth factors and orchestrate both innate and adaptive immune responses. Several of these factors have entered the clinical arena to support or augment the immune response. Moreover, the use of cytokines has recently been expanded to patients without an overtly defective immune system but who have either significant infection or infection with drug resistant organisms. The use of cytokines as adjuvants in the treatment of respiratory infections is reviewed.
Asthma is an inflammatory disease of the airways characterized by airway obstruction due to inappropriate immune responses against some environmental moieties like house dust mite (HDM) and pollens. ...Depending on the frequency and intensity of exacerbations experienced, asthma can be categorized as mild-moderate asthma (MMA) that is corticosteroid (CS)- responsive and severe asthma (SA) that is poorly managed by CS. SA constitutes only 5–10% of all asthmatics but accounts for 50% of the healthcare costs and morbidity associated with the disease. The presence of neutrophils in asthma has been correlated with refractoriness to CS, fatal exacerbations and severity. To date, our understanding of the pathological mechanisms central to neutrophil-prominent SA is minimal primarily because of the lack of a suitable animal model, limiting the development and testing of new therapeutics.
Studies in our lab have shown that unlike patients with milder disease, SA patients consistently have a mixed Th1/Th2/Th17 immune phenotype in their airways despite high dose CS treatment. With the association of viral and bacterial infections with severity and frequency of asthma exacerbations, we hypothesized that the combination of an allergen, HDM, with agents that mimic infection would induce a SA phenotype in mice. As in human severe asthmatics, the immune response in this mouse model elicits mixed granulocytic airway inflammation and airway hyperresponsiveness that are poorly responsive to CS. IFN-γ-deficient and IL-17A-deficient mice subjected to the SA model highlight the differential role played by each cytokine in modulating hallmark features of asthma such as airway inflammation, airway hyperreactivity and steroid responsiveness. To identify additional biomarkers for characterization of SA, total lung RNA from mice subjected to the SA model was analyzed by RNA-Seq. Ingenuity Pathway Analysis of the RNA-Seq data revealed potential intermediates (e.g., lipoproteins, proteases, etc.) in the IFN-γ and IL-17A signaling pathways that can be targeted for developing novel therapies for SA and potentially other steroid-refractory diseases.
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