Backbone Modification in a Protein Hydrophobic Core.

Targeted protein backbone modification can recreate tertiary structures reminiscent of folds found in nature on artificial scaffolds with improved biostability. Incorporation of altered monomers in such entities is typically limited to sites distant from the hydrophobic core to avoid potential disruptions to folding. This is limiting, as it is advantageous in some applications to incorporate artificial connectivity at buried sites. Here, we report an examination of protein backbone modification targeted specifically to hydrophobic core positions and its impacts on tertiary folded structure and fold stability. Different artificial monomer types are placed at core, core-flanking, or solvent-exposed positions in a compact three-helix protein. Effects on structure and folding energetics are assessed by NMR spectroscopy and biophysical methods. Results show that artificial residues can be well accommodated in the hydrophobic core of a defined tertiary fold, with effects on stability only modestly larger than identical changes at solvent-exposed sites. Collectively, these results provide new insights into folding behavior of protein-like artificial chains as well as strategies for the design of such molecules.

Effects of Desiccation and Freezing on Microbial Ionizing Radiation Survivability: Considerations for Mars Sample Return.

Increasingly, national space agencies are expanding their goals to include Mars exploration with sample return. To better protect Earth and its biosphere from potential extraterrestrial sources of contamination, as set forth in the Outer Space Treaty of 1967, international efforts to develop planetary protection measures strive to understand the danger of cross-contamination processes in Mars sample return missions. We aim to better understand the impact of the martian surface on microbial dormancy and survivability. Radiation resistance of microbes is a key parameter in considering survivability of microbes over geologic times on the frigid, arid surface of Mars that is bombarded by solar and galactic cosmic radiation. We tested the influence of desiccation and freezing on the ionizing radiation survival of six model microorganisms: vegetative cells of two bacteria (Deinococcus radiodurans, Escherichia coli) and a strain of budding yeast (Saccharomyces cerevisiae); and vegetative cells and endospores of three Bacillus bacteria (B. subtilis, B. megaterium, B. thuringiensis). Desiccation and freezing greatly increased radiation survival of vegetative polyploid microorganisms when applied separately, and when combined, desiccation and freezing increased radiation survival even more so. Thus, the radiation survival threshold of polyploid D. radiodurans cells can be extended from the already high value of 25 kGy in liquid culture to an astonishing 140 kGy when the cells are both desiccated and frozen. However, such synergistic radioprotective effects of desiccation and freezing were not observed in monogenomic or digenomic Bacillus cells and endospores, which are generally sterilized by 12 kGy. This difference is associated with a critical requirement for survivability under radiation, that is, repair of genome damage caused by radiation. Deinococcus radiodurans and S. cerevisiae accumulate similarly high levels of the Mn antioxidants that are required for extreme radiation resistance, as do endospores, though they greatly exceed spores in radioresistance because they contain multiple identical genome copies, which in D. radiodurans are joined by persistent Holliday junctions. We estimate ionizing radiation survival limits of polyploid DNA-based life-forms to be hundreds of millions of years of background radiation while buried in the martian subsurface. Our findings imply that forward contamination of Mars will essentially be permanent, and backward contamination is a possibility if life ever existed on Mars.

Small-Molecule Mn Antioxidants in Caenorhabditis elegans and Deinococcus radiodurans Supplant MnSOD Enzymes during Aging and Irradiation.

Denham Harman's oxidative damage theory identifies superoxide (O2•-) radicals as central agents of aging and radiation injury, with Mn2+-dependent superoxide dismutase (MnSOD) as the principal O2•--scavenger. However, in the radiation-resistant nematode Caenorhabditis elegans, the mitochondrial antioxidant enzyme MnSOD is dispensable for longevity, and in the model bacterium Deinococcus radiodurans, it is dispensable for radiation resistance. Many radiation-resistant organisms accumulate small-molecule Mn2+-antioxidant complexes well-known for their catalytic ability to scavenge O2•-, along with MnSOD, as exemplified by D. radiodurans. Here, we report experiments that relate the MnSOD and Mn-antioxidant content to aging and oxidative stress resistances and which indicate that C. elegans, like D. radiodurans, may rely on Mn-antioxidant complexes as the primary defense against reactive oxygen species (ROS). Wild-type and ΔMnSOD D. radiodurans and C. elegans were monitored for gamma radiation sensitivities over their life spans while gauging Mn2+-antioxidant content by electron paramagnetic resonance (EPR) spectroscopy, a powerful new approach to determining the in vivo Mn-antioxidant content of cells as they age. As with D. radiodurans, MnSOD is dispensable for radiation survivability in C. elegans, which hyperaccumulates Mn-antioxidants exceptionally protective of proteins. Unexpectedly, ΔMnSOD mutants of both the nematodes and bacteria exhibited increased gamma radiation survival compared to the wild-type. In contrast, the loss of MnSOD renders radiation-resistant bacteria sensitive to atmospheric oxygen during desiccation. Our results support the concept that the disparate responses to oxidative stress are explained by the accumulation of Mn-antioxidant complexes which protect, complement, and can even supplant MnSOD. IMPORTANCE The current theory of cellular defense against oxidative damage identifies antioxidant enzymes as primary defenders against ROS, with MnSOD being the preeminent superoxide (O2•-) scavenger. However, MnSOD is shown to be dispensable both for radiation resistance and longevity in model organisms, the bacterium Deinococcus radiodurans and the nematode Caenorhabditis elegans. Measured by electron paramagnetic resonance (EPR) spectroscopy, small-molecule Mn-antioxidant content was shown to decline in unison with age-related decreases in cell proliferation and radioresistance, which again are independent of MnSOD presence. Most notably, the Mn-antioxidant content of C. elegans drops precipitously in the last third of its life span, which links with reports that the steady-state level of oxidized proteins increases exponentially during the last third of the life span in animals. This leads us to propose that global responses to oxidative stress must be understood through an extended theory that includes small-molecule Mn-antioxidants as potent O2•--scavengers that complement, and can even supplant, MnSOD.

Characterization of unique functionalities in c-Src domains required for osteoclast podosome belt formation.

Deletion of c-Src, a ubiquitously expressed tyrosine kinase, results in osteoclast dysfunction and osteopetrosis, in which bones harden into "stone." In contrast, deletion of the genes encoding other members of the Src family kinase (SFK) fails to produce an osteopetrotic phenotype. This suggests that c-Src performs a unique function in the osteoclast that cannot be compensated for by other SFKs. We aimed to identify the molecular basis of this unique role in osteoclasts and bone resorption. We found that c-Src, Lyn, and Fyn were the most highly expressed SFKs in WT osteoclasts, whereas Hck, Lck, Blk, and Fgr displayed low levels of expression. Formation of the podosome belt, clusters of unique actin assemblies, was disrupted in src-/- osteoclasts; introduction of constitutively activated SFKs revealed that only c-Src and Fyn could restore this process. To identify the key structural domains responsible, we constructed chimeric Src-Hck and Src-Lyn constructs in which the unique, SH3, SH2, or catalytic domains had been swapped. We found that the Src unique, SH3, and kinase domains were each crucial to establish Src functionality. The SH2 domain could however be substituted with Lyn or Hck SH2 domains. Furthermore, we demonstrate that c-Src's functionality is, in part, derived from an SH3-proximal proline-rich domain interaction with c-Cbl, leading to phosphorylation of c-Cbl Tyr700. These data help clarify Src's unique functionality in the organization of the cytoskeleton in osteoclasts, required for efficient bone resorption and explain why c-Src cannot be replaced, in osteoclasts, by other SFKs.

High-dimensional profiling clusters asthma severity by lymphoid and non-lymphoid status.

Clinical definitions of asthma fail to capture the heterogeneity of immune dysfunction in severe, treatment-refractory disease. Applying mass cytometry and machine learning to bronchoalveolar lavage (BAL) cells, we find that corticosteroid-resistant asthma patients cluster largely into two groups: one enriched in interleukin (IL)-4+ innate immune cells and another dominated by interferon (IFN)-γ+ T cells, including tissue-resident memory cells. In contrast, BAL cells of a healthier population are enriched in IL-10+ macrophages. To better understand cellular mediators of severe asthma, we developed the Immune Cell Linkage through Exploratory Matrices (ICLite) algorithm to perform deconvolution of bulk RNA sequencing of mixed-cell populations. Signatures of mitosis and IL-7 signaling in CD206-FcεRI+CD127+IL-4+ innate cells in one patient group, contrasting with adaptive immune response in T cells in the other, are preserved across technologies. Transcriptional signatures uncovered by ICLite identify T-cell-high and T-cell-poor severe asthma patients in an independent cohort, suggesting broad applicability of our findings.

Acquired mutations and transcriptional remodeling in long-term estrogen-deprived locoregional breast cancer recurrences.

BACKGROUND: Endocrine therapy resistance is a hallmark of advanced estrogen receptor (ER)-positive breast cancer. In this study, we aimed to determine acquired genomic changes in endocrine-resistant disease. METHODS: We performed DNA/RNA hybrid-capture sequencing on 12 locoregional recurrences after long-term estrogen deprivation and identified acquired genomic changes versus each tumor's matched primary. RESULTS: Despite being up to 7 years removed from the primary lesion, most recurrences harbored similar intrinsic transcriptional and copy number profiles. Only two genes, AKAP9 and KMT2C, were found to have single nucleotide variant (SNV) enrichments in more than one recurrence. Enriched mutations in single cases included SNVs within transcriptional regulators such as ARID1A, TP53, FOXO1, BRD1, NCOA1, and NCOR2 with one local recurrence gaining three PIK3CA mutations. In contrast to DNA-level changes, we discovered recurrent outlier mRNA expression alterations were common-including outlier gains in TP63 (n = 5 cases [42%]), NTRK3 (n = 5 [42%]), NTRK2 (n = 4 [33%]), PAX3 (n = 4 [33%]), FGFR4 (n = 3 [25%]), and TERT (n = 3 [25%]). Recurrent losses involved ESR1 (n = 5 [42%]), RELN (n = 5 [42%]), SFRP4 (n = 4 [33%]), and FOSB (n = 4 [33%]). ESR1-depleted recurrences harbored shared transcriptional remodeling events including upregulation of PROM1 and other basal cancer markers. CONCLUSIONS: Taken together, this study defines acquired genomic changes in long-term, estrogen-deprived disease; highlights the importance of longitudinal RNA profiling; and identifies a common ESR1-depleted endocrine-resistant breast cancer subtype with basal-like transcriptional reprogramming.

Biliary-Atresia-Associated Mannosidase-1-Alpha-2 Gene Regulates Biliary and Ciliary Morphogenesis and Laterality.

BACKGROUND/AIMS: Infectious and genetic factors are invoked, respectively in isolated biliary atresia (BA), or syndromic BA, with major extrahepatic anomalies. However, isolated BA is also associated with minor extrahepatic gut and cardiovascular anomalies and multiple susceptibility genes, suggesting common origins. METHODS: We investigated novel susceptibility genes with genome-wide association, targeted sequencing and tissue staining in BA requiring liver transplantation, independent of BA subtype. Candidate gene effects on morphogenesis, developmental pathways, and ciliogenesis, which regulates left-right patterning were investigated with zebrafish knockdown and mouse knockout models, mouse airway cell cultures, and liver transcriptome analysis. RESULTS: Single nucleotide polymorphisms in Mannosidase-1-α-2 (MAN1A2) were significantly associated with BA and with other polymorphisms known to affect MAN1A2 expression but were not differentially enriched in either BA subtype. In zebrafish embryos, man1a2 knockdown caused poor biliary network formation, ciliary dysgenesis in Kupffer's vesicle, cardiac and liver heterotaxy, and dysregulated egfra and other developmental genes. Suboptimal man1a2 knockdown synergized with suboptimal EGFR signaling or suboptimal knockdown of the EGFR pathway gene, adenosine-ribosylation-factor-6, which had minimal effects individually, to reproduce biliary defects but not heterotaxy. In cultured mouse airway epithelium, Man1a2 knockdown arrested ciliary development and motility. Man1a2 -/- mice, which experience respiratory failure, also demonstrated portal and bile ductular inflammation. Human BA liver and Man1a2 -/- liver exhibited reduced Man1a2 expression and dysregulated ciliary genes, known to cause multisystem human laterality defects. CONCLUSION: BA requiring transplantation associates with sequence variants in MAN1A2. man1a2 regulates laterality, in addition to hepatobiliary morphogenesis, by regulating ciliogenesis in zebrafish and mice, providing a novel developmental basis for multisystem defects in BA.

Interleukin-17-induced neutrophil extracellular traps mediate resistance to checkpoint blockade in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) remains a lethal malignancy with an immunosuppressive microenvironment that is resistant to most therapies. IL17 is involved in pancreatic tumorigenesis, but its role in invasive PDAC is undetermined. We hypothesized that IL17 triggers and sustains PDAC immunosuppression. We inhibited IL17/IL17RA signaling using pharmacological and genetic strategies alongside mass cytometry and multiplex immunofluorescence techniques. We uncovered that IL17 recruits neutrophils, triggers neutrophil extracellular traps (NETs), and excludes cytotoxic CD8 T cells from tumors. Additionally, IL17 blockade increases immune checkpoint blockade (PD-1, CTLA4) sensitivity. Inhibition of neutrophils or Padi4-dependent NETosis phenocopies IL17 neutralization. NMR spectroscopy revealed changes in tumor lactate as a potential early biomarker for IL17/PD-1 combination efficacy. Higher expression of IL17 and PADI4 in human PDAC corresponds with poorer prognosis, and the serum of patients with PDAC has higher potential for NETosis. Clinical studies with IL17 and checkpoint blockade represent a novel combinatorial therapy with potential efficacy for this lethal disease.

Differential gene expression in peripheral blood mononuclear cells from children immunized with inactivated influenza vaccine.

The human immune response to inactivated influenza vaccine is dynamic and impacted by age and preexisting immunity. Our goal was to identify postvaccination transcriptomic changes in peripheral blood mononuclear cells from children. Blood samples were obtained before and at 3 or 7 days postvaccination with 2016-2017 quadrivalent inactivated influenza vaccine and RNA sequencing was performed. There were 1,466 differentially expressed genes (DEGs) for the Day 0-Day 3 group and 513 DEGs for the Day 0-Day 7 group. Thirty-three genes were common between the two groups. The majority of the transcriptomic changes at Day 3 represented innate inflammation and apoptosis pathways. Day 7 DEGs were characterized by activation of cellular processes, including the regulation of cytoskeleton, junctions, and metabolism, and increased expression of immunoglobulin genes. DEGs at Day 3 were compared between older and younger children revealing increased inflammatory gene expression in the older group. Vaccine history in the year prior to the study was characterized by robust DEGs at Day 3 with decreased phagosome and dendritic cell maturation in those who had been vaccinated in the previous year. PBMC responses to inactivated influenza vaccination in children differed significantly by the timing of sampling, patient age, and vaccine history. These data provide insight into the expected molecular pathways to be temporally altered by influenza vaccination in children.

FSTL-1 Attenuation Causes Spontaneous Smoke-Resistant Pulmonary Emphysema.

Rationale: The role of FSTL-1 (follistatin-like 1) in lung homeostasis is unknown.Objectives: We aimed to define the impact of FSTL-1 attenuation on lung structure and function and to identify FSTL-1-regulated transcriptional pathways in the lung. Further, we aimed to analyze the association of FSTL-1 SNPs with lung disease.Methods: FSTL-1 hypomorphic (FSTL-1 Hypo) mice underwent lung morphometry, pulmonary function testing, and micro-computed tomography. Fstl1 expression was determined in wild-type lung cell populations from three independent research groups. RNA sequencing of wild-type and FSTL-1 Hypo mice identified FSTL-1-regulated gene expression, followed by validation and mechanistic in vitro examination. FSTL1 SNP analysis was performed in the COPDGene (Genetic Epidemiology of Chronic Obstructive Pulmonary Disease) cohort.Measurements and Main Results: FSTL-1 Hypo mice developed spontaneous emphysema, independent of smoke exposure. Fstl1 is highly expressed in the lung by mesenchymal and endothelial cells but not immune cells. RNA sequencing of whole lung identified 33 FSTL-1-regulated genes, including Nr4a1, an orphan nuclear hormone receptor that negatively regulates NF-κB (nuclear factor-κB) signaling. In vitro, recombinant FSTL-1 treatment of macrophages attenuated NF-κB p65 phosphorylation in an Nr4a1-dependent manner. Within the COPDGene cohort, several SNPs in the FSTL1 region corresponded to chronic obstructive pulmonary disease and lung function.Conclusions: This work identifies a novel role for FSTL-1 protecting against emphysema development independent of smoke exposure. This FSTL-1-deficient emphysema implicates regulation of immune tolerance in lung macrophages through Nr4a1. Further study of the mechanisms involving FSTL-1 in lung homeostasis, immune regulation, and NF-κB signaling may provide additional insight into the pathophysiology of emphysema and inflammatory lung diseases.

Biomarkers that differentiate false positive urinalyses from true urinary tract infection.

BACKGROUND: The specificity of the leukocyte esterase test (87%) is suboptimal. The objective of this study was to identify more specific screening tests that could reduce the number of children who unnecessarily receive antimicrobials to treat a presumed urinary tract infection (UTI). METHODS: Prospective cross-sectional study to compare inflammatory proteins in blood and urine samples collected at the time of a presumptive diagnosis of UTI. We also evaluated serum RNA expression in a subset. RESULTS: We enrolled 200 children; of these, 89 were later demonstrated not to have a UTI based on the results of the urine culture obtained. Urinary proteins that best discriminated between children with UTI and no UTI were involved in T cell response proliferation (IL-9, IL-2), chemoattractants (CXCL12, CXCL1, CXCL8), the cytokine/interferon pathway (IL-13, IL-2, INFγ), or involved in innate immunity (NGAL). The predictive power (as measured by the area under the curve) of a combination of four urinary markers (IL-2, IL-9, IL-8, and NGAL) was 0.94. Genes in the pathways related to inflammation were also upregulated in serum of children with UTI. CONCLUSIONS: Urinary proteins involved in the inflammatory response may be useful in identifying children with false positive results with current screening tests for UTI; this may reduce unnecessary treatment.

Intestinal IL-17R Signaling Constrains IL-18-Driven Liver Inflammation by the Regulation of Microbiome-Derived Products.

Interleukin (IL)-17 signaling to the intestinal epithelium regulates the intestinal microbiome. Given the reported links between intestinal dysbiosis, bacterial translocation, and liver disease, we hypothesize that intestinal IL-17R signaling plays a critical role in mitigating hepatic inflammation. To test this, we study intestinal epithelium-specific IL-17RA-deficient mice in an immune-driven hepatitis model. At the naive state, these mice exhibit microbiome dysbiosis and increased translocation of bacterial products (CpG DNA), which drives liver IL-18 production. Upon disease induction, absence of enteric IL-17RA signaling exacerbates hepatitis and hepatocyte cell death. IL-18 is necessary for disease exacerbation and is associated with increased activated hepatic lymphocytes based on Ifng and Fasl expression. Thus, intestinal IL-17R regulates translocation of TLR9 ligands and constrains susceptibility to hepatitis. These data connect enteric Th17 signaling and the microbiome in hepatitis, with broader implications on the effects of impaired intestinal immunity and subsequent release of microbial products observed in other extra-intestinal pathologies.

Transcriptomic and Proteomic Approaches to Finding Novel Diagnostic and Immunogenic Candidates in Pneumocystis.

Pneumocystis pneumonia is the most common serious opportunistic infection in patients with HIV/AIDS. Furthermore, Pneumocystis pneumonia is a feared complication of the immunosuppressive drug regimens used to treat autoimmunity, malignancy, and posttransplantation rejection. With an increasing at-risk population, there is a strong need for novel approaches to discover diagnostic and vaccine targets. There are multiple challenges to finding these targets, however. First, Pneumocystis has a largely unannotated genome. To address this, we evaluated each protein encoded within the Pneumocystis genome by comparisons to proteins encoded within the genomes of other fungi using NCBI BLAST. Second, Pneumocystis relies on a multiphasic life cycle, as both the transmissible form (the ascus) and the replicative form (the trophozoite [troph]) reside within the alveolar space of the host. To that end, we purified asci and trophs from Pneumocystis murina and utilized transcriptomics to identify differentially regulated genes. Two such genes, Arp9 and Sp, are differentially regulated in the ascus and the troph, respectively, and can be utilized to characterize the state of the Pneumocystis life cycle in vivoGsc1, encoding a ß-1,3-glucan synthase with a large extracellular domain previously identified using surface proteomics, was more highly expressed on the ascus form of Pneumocystis GSC-1 ectodomain immunization generated a strong antibody response that demonstrated the ability to recognize the surface of the Pneumocystis asci. GSC-1 ectodomain immunization was also capable of reducing ascus burden following primary challenge with Pneumocystis murina Finally, mice immunized with the GSC-1 ectodomain had limited fungal burden following natural transmission of Pneumocystis using a cohousing model.IMPORTANCE The current report enhances our understanding of Pneumocystis biology in a number of ways. First, the current study provided a preliminary annotation of the Pneumocystis murina genome, addressing a long-standing issue in the field. Second, this study validated two novel transcripts enriched in the two predominant life forms of Pneumocystis These findings allow better characterization of the Pneumocystis life cycle in vivo and could be valuable diagnostic tools. Furthermore, this study outlined a novel pipeline of -omics techniques capable of revealing novel antigens (e.g., GSC-1) for the development of vaccines against Pneumocystis.

A method of processing nasopharyngeal swabs to enable multiple testing.

OBJECTIVE: To develop a method to perform multiple tests on a single nasopharyngeal (NP) swab. METHODS: We collected a NP swab on children aged 2-12 years with acute sinusitis and processed it for bacterial culture, viruses, cytokine expression, and 16S ribosomal RNA gene sequencing analysis. During the course of the study, we expand the scope of evaluation to include RNA-sequencing, which we accomplished by cutting the tip of the swab. RESULTS: Of the 174 children enrolled, 126 (72.4%) had a positive bacterial culture and 121 (69.5%) tested positive for a virus. Cytokine measurement, as judged by adequate levels of a housekeeping enzyme (glyceraldehyde 3-phosphate dehydrogenase), appeared successful. From the samples used for 16S ribosomal sequencing we recovered, on average, 16,000 sequences per sample, accounting for a total of 2646 operational taxonomic units across all samples sequenced. Samples used for RNA-sequencing had a mean RNA integrity number of 6.0. Cutting the tip of the swab did not affect the recovery yield for viruses or bacteria, nor did it affect species richness in microbiome analysis. CONCLUSION: We describe a minimally invasive sample collection protocol that allows for multiple diagnostic and research investigations in young children.

Host and Bacterial Markers that Differ in Children with Cystitis and Pyelonephritis.

OBJECTIVE: To determine whether treatment for urinary tract infections in children could be individualized using biomarkers for acute pyelonephritis. STUDY DESIGN: We enrolled 61 children with febrile urinary tract infections, collected blood and urine samples, and performed a renal scan within 2 weeks of diagnosis to identify those with pyelonephritis. Renal scans were interpreted centrally by 2 experts. We measured inflammatory proteins in blood and urine using LUMINEX or an enzyme-linked immunosorbent assay. We evaluated serum RNA expression using RNA sequencing in a subset of children. Finally, for children with Escherichia coli isolated from urine cultures, we performed a polymerase chain reaction for 4 previously identified virulence genes. RESULTS: Urinary markers that best differentiated pyelonephritis from cystitis included chemokine (C-X-C motif) ligand (CXCL)1, CXCL9, CXCL12, C-C motif chemokine ligand 2, INF γ, and IL-15. Serum procalcitonin was the best serum marker for pyelonephritis. Genes in the interferon-γ pathway were upregulated in serum of children with pyelonephritis. The presence of E coli virulence genes did not correlate with pyelonephritis. CONCLUSIONS: Immune response to pyelonephritis and cystitis differs quantitatively and qualitatively; this may be useful in differentiating these 2 conditions.

Inflammatory Mediator Expression Associated With Antibody Response Induced by Live Attenuated vs Inactivated Influenza Virus Vaccine in Children.

BACKGROUND: The reasons for differences in vaccine effectiveness between live attenuated influenza vaccine (LAIV) and inactivated influenza vaccine (IIV) are not clear. METHODS: Blood samples were obtained before vaccination and at days 7 and 21 postvaccination with 2015-2016 quadrivalent IIV or LAIV. Serologic response to the vaccine was measured by hemagglutination inhibition assay. Targeted RNA sequencing and serum cytokine analysis were performed. Paired analyses were used to determine gene expression and were compared between IIV and LAIV recipients. Classification And Regression Trees analysis (CART) identified the strongest associations with vaccine response. RESULTS: Forty-six enrollees received IIV, and 25 received LAIV. The mean age was 11.5 (±3.7) years. Seroconversion with IIV was associated with changes in expression of PRKRA and IFI6. Nonseroconversion for both IIV and LAIV was characterized by increased interferon-stimulated gene expression. Seroprotection with both vaccines was associated with altered expression of CXCL2 and CD36. For LAIV, CART showed that changes in expression of CD80, CXCL2, and CASP1 were associated with seroprotection. Serum cytokines showed that IIV seroconversion was associated with decreased CCL3. LAIV seroprotection tracked with decreased tumor necrosis factor-α and interferon-γ. CONCLUSIONS: Distinct markers of seroconversion and seroprotection against IIV and LAIV were identified using immunophenotyping and CART analysis.

Murine models of Pneumocystis infection recapitulate human primary immune disorders.

Despite the discovery of key pattern recognition receptors and CD4+ T cell subsets in laboratory mice, there is ongoing discussion of the value of murine models to reflect human disease. Pneumocystis is an AIDS-defining illness, in which risk of infection is inversely correlated with peripheral CD4+ T cell counts. Due to medical advances in the control of HIV, the current epidemiology of Pneumocystis infection is predominantly due to primary human immunodeficiencies and immunosuppressive therapies. To this end, we found that every human genetic immunodeficiency associated with Pneumocystis infection that has been tested in mice recapitulated susceptibility. For example, humans with a loss-of-function IL21R mutation are severely immunocompromised. We found that IL-21R, in addition to CD4+ T cell intrinsic STAT3 signaling, were required for generating protective antifungal class-switched antibody responses, as well as effector T cell-mediated protection. Furthermore, CD4+ T cell intrinsic IL-21R/STAT3 signaling was required for CD4+ T cell effector responses, including IL-22 production. Recombinant IL-22 administration to Il21r-/- mice induced the expression of a fungicidal peptide, cathelicidin antimicrobial peptide, which showed in vitro fungicidal activity. In conclusion, SPF laboratory mice faithfully replicate many aspects of human primary immunodeficiency and provide useful tools to understand the generation and nature of effector CD4+ T cell immunity.

Immune Cell Production of Interleukin 17 Induces Stem Cell Features of Pancreatic Intraepithelial Neoplasia Cells.

BACKGROUND & AIMS: Little is known about how the immune system affects stem cell features of pancreatic cancer cells. Immune cells that produce interleukin 17A (IL17A) in the chronically inflamed pancreas (chronic pancreatitis) contribute to pancreatic interepithelial neoplasia (PanIN) initiation and progression. We investigated the effects that IL17A signaling exerts on pancreatic cancer progenitor cells and the clinical relevance of this phenomena. METHODS: We performed studies with Mist1Cre;LSLKras;Rosa26mTmG (KCiMist;G) and Kras(G12D);Trp53(R172H);Pdx1-Cre (KPC) mice (which upon tamoxifen induction spontaneously develop PanINs) and control littermates. Some mice were injected with neutralizing antibodies against IL17A or control antibody. Pancreata were collected, PanIN epithelial cells were isolated by flow cytometry based on lineage tracing, and gene expression profiles were compared. We collected cells from pancreatic tumors of KPC mice, incubated them with IL17 or control media, measured expression of genes regulated by IL17 signaling, injected the cancer cells into immune competent mice, and measured tumor growth. IL17A was overexpressed in pancreata of KCiMist mice from an adenoviral vector. Pancreata were collected from all mice and analyzed by histology and immunohistochemistry. Levels of DCLK1 and other proteins were knocked down in KPC pancreatic cancer cells using small interfering or short hairpin RNAs; cells were analyzed by immunoblotting. We obtained 65 pancreatic tumor specimens from patients, analyzed protein levels by immunohistochemistry, and compared results with patient survival times. We also analyzed gene expression levels and patient outcome using The Cancer Genome Atlas database. RESULTS: PanIN cells from KCiMist;G mice had a gene expression pattern associated with embryonic stem cells. Mice given injections of IL17-neutralizing antibodies, or with immune cells that did not secrete IL17, lost this expression pattern and had significantly decreased expression of DCLK1 and POU2F3, which regulate tuft cell development. KCiMist mice that overexpressed IL17 formed more PanINs, with more DCLK1-positive cells, than control mice. Pancreatic tumor cells from KPC mice and human Capan-2 cells exposed to IL17A had increased activation of NF-κB and mitogen-activated protein kinase signaling and increased expression of DCLK1 and ALDH1A1 (a marker of embryonic stem cells) compared with cells in control media. These cells also formed tumors faster that cells not exposed to IL17 when they were injected into immunocompetent mice. KPC cells with knockdown of DCLK1 expressed lower levels of ALDH1A1 after incubation with IL17 than cells without knockdown. Expression of the IL17 receptor C was higher in DCLK1-positive PanIN cells from mice compared with DCLK1-negative PanIN cells. In human pancreatic tumor tissues, high levels of DCLK1 associated with a shorter median survival time of patients (17.7 months, compared with 26.6 months of patients whose tumors had low levels of DCLK1). Tumor levels of POU2F3 and LAMC2 were also associated with patient survival time. CONCLUSIONS: In studies of mouse and human pancreatic tumors and precursors, we found that immune cell-derived IL17 regulated development of tuft cells and stem cell features of pancreatic cancer cells via increased expression of DCLK1, POU2F3, ALDH1A1, and IL17RC. Strategies to disrupt this pathway might be developed to prevent pancreatic tumor growth and progression.

Differential gene expression elicited by children in response to the 2015-16 live attenuated versus inactivated influenza vaccine.

BACKGROUND: In recent influenza seasons, the live attenuated influenza vaccine (LAIV) has not demonstrated the same level of vaccine effectiveness as that observed among children who received the inactivated influenza vaccine (IIV). To better understand this difference, this study compared the mRNA sequencing transcription profile (RNA seq) in children who received either IIV or LAIV. METHODS: Children 3-17years of age receiving quadrivalent influenza vaccine were enrolled. Blood samples were collected on Day 0 prior to vaccination and again on Day 7 (range 6-10days) following vaccination. Total RNA was isolated from PAXgene tubes and sequenced for a custom panel of 89 transcripts using the TruSeq Targeted RNA Expression method. Fold differences in normalized RNA seq counts from Day 0 to Day 7 were calculated, log2 transformed and compared between the two vaccine groups. RESULTS: Of 72 children, 46 received IIV and 26 received LAIV. Following IIV vaccination, 7 genes demonstrated significant differential expression at Day 7 (down-regulated). In contrast, following LAIV vaccination, 8 genes demonstrated significant differential expression at Day 7 (5 up-regulated and 3 down-regulated). Only two genes demonstrated similar patterns of regulation in both groups. CONCLUSIONS: Differential regulation of genes was observed between 2015-16 LAIV and IIV recipients. These results help to elucidate the immune response to influenza vaccines and may be related to the difference in vaccine effectiveness observed in recent years between LAIV and IIV.

Interleukin-17 limits hypoxia-inducible factor 1α and development of hypoxic granulomas during tuberculosis.

Mycobacterium tuberculosis (Mtb) is a global health threat, compounded by the emergence of drug-resistant strains. A hallmark of pulmonary tuberculosis (TB) is the formation of hypoxic necrotic granulomas, which upon disintegration, release infectious Mtb. Furthermore, hypoxic necrotic granulomas are associated with increased disease severity and provide a niche for drug-resistant Mtb. However, the host immune responses that promote the development of hypoxic TB granulomas are not well described. Using a necrotic Mtb mouse model, we show that loss of Mtb virulence factors, such as phenolic glycolipids, decreases the production of the proinflammatory cytokine IL-17 (also referred to as IL-17A). IL-17 production negatively regulates the development of hypoxic TB granulomas by limiting the expression of the transcription factor hypoxia-inducible factor 1α (HIF1α). In human TB patients, HIF1α mRNA expression is increased. Through genotyping and association analyses in human samples, we identified a link between the single nucleotide polymorphism rs2275913 in the IL-17 promoter (-197G/G), which is associated with decreased IL-17 production upon stimulation with Mtb cell wall. Together, our data highlight a potentially novel role for IL-17 in limiting the development of hypoxic necrotic granulomas and reducing disease severity in TB.