Autophagy involving core machinery proteins such as ATG9A is part of a cytoprotective process whose dysregulation is associated with carcinogenesis, neurodegeneration and autoimmunity, but few ...examples exist of monogenic diseases to further insights into human disease. In the present studies, we report a patient with novel compound heterozygous mutations in ATG9A after patient and parental DNA were subjected to whole exome sequencing. The patient developed hyperplastic proliferations of T and B cells in lung and brain and exhibited defects in lymphocyte memory cell populations after developing an infection with Epstein-Barr virus (EBV). Peripheral blood leukocytes from the patient exhibited defects in autophagic activity and EBV-transformed cells redemonstrated this defect which was also associated with defective NFKB (nuclear factor kappa B) signaling and accumulation of the pro-proliferative protein MYC (MYC proto-oncogene, bHLH transcription factor) and anti-apoptotic protein BCL2 (BCL2 apoptosis regulator), leading to increased proliferative capacity. Defects were corrected after transformation with plasmids expressing ATG9A and after treatment of cells as well as the patient with the MTOR (mechanistic target of rapamycin kinase) inhibitor, autophagy-inducing immunosuppressant rapamycin. These results point to a novel role of ATG9A and autophagy in human cellular signaling associated with hyperplasia and lymphocyte biology and provide an example how genetic studies may suggest effective specific therapeutic interventions.
BCL2: BCL2 apoptosis regulator; BCL10: BCL10 immune signaling adaptor; CARD11: caspase recruitment domain family member 11; CBM: CARD11-BCL10-MALT1; CR2: complement C3d receptor 2; EBNA: Epstein Barr nuclear antigen; EBV: Epstein-Barr virus; FCGR3A; Fc gamma receptor IIIa; GLILD: granulomatous-lymphocytic interstitial lung disease; HV: healthy volunteer; IKBKB/IKB kinase: inhibitor of nuclear factor kappa B kinase subunit beta; IL2RA: interleukin 2 receptor subunit alpha; MALT1: MALT1 paracaspase; MS4A1: membrane spanning 4-domain A1; MTOR: mechanistic target of rapamycin kinase; MYC: MYC proto-oncogene, bHLH: transcription factor; NCAM1: neural cell adhesion molecule 1; NFKB: nuclear factor kappa B; NIAID: National Institute of Allergy and Infectious Diseases; NK: natural killer; PTPRC: protein tyrosine phosphatase receptor type C; SELL: selectin L; PBMCs: peripheral blood mononuclear cells; TR: T cell receptor; Tregs: regulatory T cells; WT: wild-type
Abstract
IL-17A and IL-22 derived from CD4+ T cells are significant for both inflammatory and tissue protective responses. However, it remains unclear whether IL-17A and IL-22 can be regulated ...differently to achieve targeted functional outcome.
Here, we showed that Nrf2 selectively regulated IL-17A and IL-22 responses in CD4+ T cells. Nrf2−/− mice challenged with Ovalbumin (Ova) + LPS displayed reduced IL-22 but enhanced IL-17A response. Interestingly, CDDO-Im, a selective Nrf2 activator, induced IL-22 but suppressed IL-17A response in CD4+ T cells polarized under Th17 cell condition. CDDO-Im-activated CD4+ T cells had lower Rorc, but increased Cybb, Sod1 and Sod3 transcript. Luciferase reporter assay showed that IL-17A promoter activity is inhibited by Nrf2 at the presence of RORγT, suggesting that inhibition of IL-17A by Nrf2 is RORγT-dependent. CDDO-Im-impaired IL-17A response was also abolished in SOD3−/−, showing that SOD3 is involved in CDDO-Im-mediated inhibition of IL-17A. Furthermore, we found that CDDO-Im induced aryl hydrocarbon receptor (Ahr) and its downstream Cyp1a1 and Cyp1b1 gene expression. Additionally, CDDO-Im induced Nqo1 expression, an Nrf2-activated gene, in WT mice but not in Ahr−/− mice. CDDO-Im-mediated induction of IL-22 production in CD4+ T cells was abrogated in Ahr−/− mice, supporting that Ahr is involved in the induction of IL-22 by Nrf2. Finally, upon the stimulation of anti-CD3 and anti-CD28, CDDO-Im also mediated the inhibition of IL-17A but induction of IL-22 production in PBMCs from relapsing-remitting multiple sclerosis patients.
Collectively, our data show that Nrf2 promotes IL-22 production via Ahr, whereas RORγT and SOD3 are involved in Nrf2-mediated inhibition of IL-17A expression in Th17 cells.
IL-22-IL-22R signaling plays a crucial role in regulating host defenses against extracellular pathogens particularly in the intestine, through the induction of antimicrobial peptides and chemotactic ...genes. However, the role of IL-22- IL-22R is understudied in Spn lung infection, a prevalent pathogen of pneumonia. This paper presents the findings of IL-22 signaling during a murine model of pneumococcal pneumonia and improvement of bacterial burden upon IL-22 administration. IL-22 was rapidly induced in the lung during pneumococcal infection in wild type mice and
Il22
−/−
mice had higher pneumococcal burdens compared to controls. Additionally, mice with hepatic-specific deletion of
Il22ra1
also had higher bacterial burdens in lungs compared to littermate controls after intrapulmonary pneumococcal infection, suggesting that IL-22 signaling in the liver is important to control pneumococcal pneumonia. Thus, we hypothesized that enhancement of IL-22 signaling would control pneumococcal burden in lung tissues in an experimental pneumonia model. Administration of recombinant IL-22 systemically to infected wild type mice decreased bacterial burden in lung and liver at 24 hours post infection. Our in vitro studies also showed that mice treated with IL-22 had increased C3 expression in the liver compared to the isotype control group. Furthermore, serum from mice treated with IL-22 had improved opsonic capacity by increasing C3 binding on Spn. Taken together, endogenous IL-22 and hepatic IL-22R signaling play critical roles in controlling pneumococcal lung burden and systemic IL-22 decreases bacterial burden in the lungs and peripheral organs by potentiating C3 opsonization on bacterial surfaces, through the increase of hepatic C3 expression.
Abstract
Pneumocystis (PC) is a fungus that causes pneumonia in immunocompromised individuals and remains the most common serious opportunistic infection in individuals diagnosed with HIV. ...Furthermore, PC has re-emerged in the HIV-negative population, as patients on immunosuppressive therapies for transplantation or malignancy are increasingly susceptible to PC pneumonia. As the at-risk population increases, the discovery of conserved PC antigens could lead to the development of vaccines, enhanced diagnostic tests and monoclonal antibody therapies. To that end, we have used RNA sequencing of separated PC life forms and surface biotinylation of whole PC to identify differentially expressed surface antigens. Histone 2B (H2B) appears to be an extracellular antigen with increased expression in the cyst life form, while a GPI-anchored protein (GPI) is more abundant on the troph life form. We used DNA vaccination to assess the immunogenicity of these antigens followed by CD4 depletion and challenge with live PC. H2B vaccination resulted in a significant increase in PC-specific IgG. After PC challenge, H2B vaccinated mice had a significant half-log reduction in PC lung burden when compared to unvaccinated controls. Similarly, GPI vaccinated mice had an increase in PC- and GPI-specific IgG, while also having a half-log reduction in PC lung burden. Taken together, these results suggest that H2B and GPI are novel PC antigens with vaccine potential in need of further exploration.
Abstract
Pneumocystis pneumonia (PCP) remains one of the leading causes of death in immunocompromised patients. Disease is attributed to the loss of CD4 T cells; however recent studies suggest that B ...cells also play a protective role in clearing PC in an Ig-independent manner through antigen presentation. Several patients treated with anti-CD20 have developed PCP and thus we investigated the effect of anti-CD20 on primary PC infection. We hypothesized that B cells in part function as a critical antigen-presenting cell to helper T cells. To test this, C57BL/6 WT mice were administered anti-CD20, anti-CD4 (as a pos. control) or both, and then infected with a 105 inoculum of Pneumocystis murina. Lung mononuclear cells were harvested two weeks after infection, assessed for cellularity and then stimulated in vitro with PC antigen for cytokine profiling. Even though CD20-depleted animals had unaltered T cells numbers, there were diminished cytokine responses for IL-5, IL-17, and IL-10. These data suggest that B cells are involved in mediating optimal T cell cytokine responses against PC. We are currently testing if B cells are required in the recall responses from memory T cells by performing B cell depletion during the re-challenge of mice that have recovered from primary PC infection. We are also investigating if B cells modify T regulatory cells during Immune Reconstitution Inflammatory Syndrome (IRIS), a disease that affects a subset of patients upon reconstitution of CD4+ cells.
Abstract
Blockade or genetic deletion of IL-17RA resulted in marked increases in IL-17 response by T-cells. We hypothesize that the hyper Th17 response in IL-17 deficient mice is due to alterations ...of the gut microbiome particularly overgrowth of segmented filamentous bacteria (SFB). Our data shows that Il17ra-/- and Il17rc-/- mice have overgrowth of SFB (10 fold higher than WT.Taconic), suggesting a critical role of IL-17 signaling in SFB colonization. Higher SFB colonization in Il17ra-/- and Il17rc-/- mice results in expansion of IL-17A and IL-22 producing Th17 cells. As further evidence that this expansion was not T-cell intrinsic, we observed similar frequencies of IL-17 producing cells in WT and Il17ra-/- when naïve T-cells were polarized in vitro to Th17 cells. Furthermore, vancomycin depletion of SFB in Il17ra-/- mice resulted into fewer Th17 cells in the lamina propria and spleen, suggesting gut microflora responsible for hyper Th17 response. SFB-colony free WT mice with intact IL-17 signaling can control SFB overgrowth but IL-17 deficient mice could not following SFB inoculation. To further define the role of IL-17 in SFB colonization, we have generated SFB-free Il17ra conditional knockout (Il17rafl/flxe2a cre) mice. SFB-free Il17rafl/flxe2a cre mice are devoid of hyper Th17 responses, and SFB inoculation resulted into acquisition of hyper Th17 phenotype as observed in Il17ra-/-. Our data suggest that IL-17 signaling regulates SFB colonization and associated Th17 responses.
Abstract
Background
Pneumococcal pneumonia is the main cause of morbidity in children younger than 2 years of age and people older than 65 years old. We have published that exogenous recombinant ...Interleukin-22 (IL-22) decreases bacterial burden in lung and extrapulmonary organs in a murine model of pneumococcal pneumonia. This beneficial effect was the result of increased phagocytosis due to IL-22 induced hepatic complement component 3. IL-22, secreted by cells of the lymphoid lineage, is the ligand for IL-22 receptor (IL-22ra1) and the decoy receptor IL-22 binding protein (IL-22bp). We hypothesized that absence of IL-22bp, the negative regulator of IL-22, would increase free IL-22, thus decreasing bacterial burden upon pneumococcal pneumonia.
Methods
We infected IL-22bp knockout (IL-22bp−/−) mice and littermate controls with Streptococcus pneumoniae 106 cfu/mouse and sacrificed them at 48 hours. We assessed lung bacterial burden, flowcytometry sorting of cell lung populations to assess IL-22bp expression, RNA sequence of bronchial brushes to assess gene expression and in vivo knockdown of Lysozyme 1 in IL-22bp−/− mice.
Results
IL-22bp−/− mice had twice the amount of IL-22 in serum compared with controls at naïve state, and once infected with pneumococcus, there was higher Il22 expression in IL-22bp−/− lungs. There was 1.5 log reduction in pneumococcal burden in IL-22bp−/− mice compared with controls. Furthermore, sorting of CD45+ and in CD45− lung cells, showed higher expression of IL-22bp in alveolar and conductive airway epithelial cells at naïve state in control mice. As IL-22ra1, the site of action of IL-22, is mostly expressed in the airway epithelium, we performed RNA sequence of bronchial brushes of infected IL-22bp−/− and control mice. Our data show higher expression of secreted antimicrobial peptides genes like Lyzosyme 1 (Lyz1), Surfactant protein C, WAP four - disulfide core domain (Wfdc)18 and Wfdc12 in infected IL22bp−/− mice compared with controls. Furthermore, knocking down of Lyz1 by siRNA in vivo in IL-22bp−/− mice reversed the protective phenotype upon infection.
Conclusion
In conclusion, our data suggest that absence of IL-22bp allows a permissive environment for IL-22 activity in the lung by increasing secretion of Lysozyme 1 from the bronchial epithelium, which is advantageous to the host upon pneumococcal lung infection.
Disclosures
All authors: No reported disclosures.
Abstract
Pneumocystis Pneumonia (PCP) is a leading cause of death in individuals with AIDS or other immunocompromised conditions. We have previously shown that mice vaccinated with DNA encoding ...full-length PC antigen Kex1 have approximately a three-log reduction in PC organism burden after challenge. Full length Kex1 is poorly expressed so to improve on this we developed codon optimized vectors expressing a truncated conserved form of Kex1, mini-kexin. This also allowed investigation of a prime boost strategy since mini-kexin could be efficiently packaged into recombinant adenovirus. Mice were vaccinated with DNA encoding mini-kexin followed by boosting with adenovirus, CD4+ T cells were then depleted by GK1.5, and finally the mice were challenged with live Pneumocystis. The results showed significantly reduced PC burden in vaccinated mice. We then proceeded to generate a protein-based, E coli produced vaccine, however, this time vaccine elicited similar anti-Kex1 titers but lacked protection. We hypothesize that post-translational modification such as glycosylation could play a role in the efficacy of mini-kexin vaccination.
Cryptococcal meningoencephalitis (CM) is the major cause of infection-related neurological death, typically seen in immunocompromised patients. However, T cell-driven inflammatory response has been ...increasingly implicated in lethal central nervous system (CNS) immunopathology in human patients and murine models. Here, we report marked up-regulation of the chemokine receptor CXCR3 axis in human patients and mice with CM. CXCR3 deletion in mice improves survival, diminishes neurological deficits, and limits neuronal damage without suppressing fungal clearance. CD4
T cell accumulation and T
1 skewing are reduced in the CNS but not spleens of infected CXCR3
mice. Adoptive transfer of WT, but not CXCR3
CD4
T cells, into CXCR3
mice phenocopies the pathology of infected WT mice. Collectively, we found that CXCR3
CD4
T cells drive lethal CNS pathology but are not required for fungal clearance during CM. The CXCR3 pathway shows potential as a therapeutic target or for biomarker discovery to limit CNS inflammatory damages.
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