Gossypium hirsutum (Gh) fiber was known to have an adhesive cotton fiber middle lamella (CFML) that joins adjacent fibers into tissue-like bundles, but it was unknown whether a CFML existed in other commercially important cotton fibers. We compared the cell wall chemistry over the time course of fiber development in Gh and Gossypium barbadense (Gb), the two most important commercial cotton species, when plants were grown in parallel in a highly controlled greenhouse. Under these growing conditions, the rate of early fiber elongation and the time of onset of secondary wall deposition were similar in fibers of the two species, but as expected the Gb fiber had a prolonged elongation period and developed higher quality compared to Gh fiber. The Gb fibers had a CFML, but it was not directly required for fiber elongation because Gb fiber continued to elongate rapidly after CFML hydrolysis. For both species, fiber at seven ages was extracted with four increasingly strong solvents, followed by analysis of cell wall matrix polysaccharide epitopes using antibody-based Glycome Profiling. Together with immunohistochemistry of fiber cross-sections, the data show that the CFML of Gb fiber contained lower levels of xyloglucan compared to Gh fiber. Xyloglucan endo-hydrolase activity was also higher in Gb fiber. In general, the data provide a rich picture of the similarities and differences in the cell wall structure of the two most important commercial cotton species.Cotton fiber is an important natural textile fiber due to its exceptional length and thickness. These properties arise largely through primary and secondary cell wall synthesis. The cotton fiber of commerce is a cellulosic secondary wall surrounded by a thin cuticulated primary wall, but there were only sparse details available about the polysaccharides in the fiber cell wall of any cotton species. In addition,  Gossypium species is the world’s most important renewable textile fiber. These single-celled fibers are highly elongated and thickened seed epidermal cells, and their useful properties depend on cell walls deposited during a staged cellular differentiation program lasting about seven weeks. Plant cell walls include fibrillar cellulose and various other polysaccharides in a surrounding matrix, e.g. xyloglucan (XG) and pectins including homogalacturonan (HG), plus variable percentages of protein and sometimes lignin. In cotton fiber, a thin primary cell wall is synthesized while the fiber achieves its extraordinary length of ∼2.5 to 3.5 cm. During the transition to secondary wall cellulose synthesis, primary wall remodeling occurs and a thin intermediary cell wall “winding” layer  is deposited. The transition stage ends when secondary wall thickening begins via the deposition of nearly pure cellulose . At harvest, the cotton fiber is composed of a thick cellulosic secondary wall that is surrounded by a ∼200 nm thick cuticulated primary wall on the fiber perimeter Cotton fiber harvested from Gossypium hirsutum (hereafter Gh) and Gossypium barbadense (Gb). Polyploidization about 1–2 million years ago unified ancestral diploid A and D genomes to form the progenitors of modern allotetraploid Gh and Gb cotton, followed by independent evolution and then domestication of the two species G. hirsutum, or Upland cotton, accounts for the majority of current cotton fiber production because of its high yield and wider environmental adaptability. However, Gb cotton is grown in selected environments because its fiber  is longer, stronger, and finer (having less mass per unit length), which makes it preferred for spinning the stronger and silkier yarns that can be woven into luxury cotton clothing. A major goal of modern cotton breeding and biotechnology is to determine the controls of these superior fiber properties in Gb, followed by selective transfer of improved fiber traits to Gh without compromising its high yield and environmental adaptability.Despite similarities in these basic cotton fiber characteristics, there are important differences in the fiber produced by the two commercial allotetraploid species, Gb fiber has a prolonged elongation phase that generates its longer fiber compared to GhGb correlates with less cellulose per unit of fiber length  Gh fiber contained epitopes typically found in primary cell matrix polysaccharides, including XG with some fucosylated epitopes, and HG with relatively low or no esterification.  and Gb cv. Phytogen 800 (Gb) were grown in parallel between April to October in an air conditioned greenhouse with a 26/22°C diurnal temperature cycle and natural lighting. Flowers were tagged on the day of anthesis so that bolls of known DPA could be collected. Details of cotton growth conditions and procedures for hand-measurement of fiber growth parameters were described previously Plants of Gheif5 . The monoclonal antibody against ß-1,3-glucan, LAMP2HI2H7 (abbreviated here as LAMP), was obtained from Biosupplies Australia  where additional information currently available for each antibody can be found.ELISA using 149 monoclonal antibodies in 21 clades  was produced and then extracted with a series of increasingly strong solvents to remove cell wall polysaccharides sequentially according to their strength of binding within the cell wall To begin the Glycome Profiling, bolls of typical size from Successive extractions for each sample  occurred starting with 50 mM ammonium oxalate . After centrifugation , the supernatant was saved at 4°C awaiting dialysis, and the pellet was successively extracted in 50 mM sodium carbonate (pH 10) containing 0.5% (w/v) sodium borohydride followed by 1 M KOH and then 4 M KOH, both containing 1.0% (w/v) sodium borohydride. (The pellets were washed with deionized water before sodium carbonate and 1 M KOH extractions). Supernatants from both KOH extractions were treated with 2–3 drops of n-octyl alcohol (to minimize frothing) and neutralized on ice with glacial acetic acid. The four supernatants were dialyzed for 48 h against de-ionized water (1∶60 sample:water ratio) with slow stirring (water replaced every 8 to 12 h).The amount of sugar in the extracts was determined by the phenol-sulfuric acid method Gh and Gb, seeds with attached fibers were harvested at 10, 19, 24, and 30 DPA from bolls of three plants and assayed in parallel. Fibers were left attached to seeds in order to replicate the “fiber straightening” protocol commonly used for fiber measurements http://www.emdchemicals.com). The supernatant was clarified by centrifugation  and adjusted to 15 ml with water containing 0.02% (w/v) NaN3 to compensate for evaporation. Standard methods of sugar and protein quantitation showed that there was 95–97% sugar and 3 to 5% protein in the extracts at different DPA (unpublished data), indicating that the hot acidic water mainly extracted cell wall carbohydrate materials from whole fibers still attached to seeds. Colorimetric immuno-dot-assays were performed as described http://carestream.com). Samples were diluted if necessary so that the intensity values were within a linear range established through using CCRC-M1 as a probe for a dilution series of 10 DPA fiber extract (unpublished data). Significant differences between means for intensity/µg sugar for the two species at each DPA were determined by T test.CCRC-M1 and CCRC-M58 were used as probes in this assay. For both Gb at 10, 17, 19, and 24 DPA were examined for the presence or absence of the CFML in the TEM as previously described Gh and Gb, cell wall epitopes in 250 nm thick cross-sections of 10, 17, and 24 DPA fibers were analyzed by fluorescence immunolabeling as described Fibers of g, 5 min then 10 min), which was stored on ice prior to assay. Activity of XG endo-hydrolase (XEH) was assayed as described with minor modification 2SO4 and 200 µl potassium tri-iodide reagent [1.0% KI (w/v) and 0.5% iodine (w/v) in water], including samples with 10 to 100 µg XG as standards. XEH activity in the fiber extracts (ng XG degraded h−1 mg protein−1) was calculated based on the decrease in the blue-green color (OD620 nm) that arises from iodine complexed with higher molecular weight XG (>10 kDa) Fiber (∼0.08 to 0.12 g fresh weight) was vortexed in 1.0 ml of 100 mM sodium acetate buffer (pH 4.0) containing 0.5 M NaCl, extracted , and centrifuged to clarify and separate the supernatant  that are apparently orthologous to the three CESA genes in Arabidopsis thaliana  that are required for secondary wall thickening Gh fiber grown under the same conditions measured  A and 1Bmeasured . The genGh and Gb, respectively. At 22 DPA, elongation in Gh fiber essentially ended, but rapid elongation in Gb fiber continued for 8 more days until 30 DPA followed by slower elongation until at least 35 DPA.The fiber of both species showed similar elongation rates between 10 to 22 days post anthesis (DPA) . The sloCESA genes were up-regulated in Gh and Gb fiber beginning at ∼17 DPA (Gb fiber.) This transition stage of fiber development includes primary cell wall remodeling and the synthesis of the winding cell wall layer, and it ends at the time that nearly 100% cellulose synthesis begins . Therefore, a similar amount of fiber was deposited on each seed in both species, but fiber weight/seed does not reveal differences that occur within individual fibers. When the fiber weight/length ratio was calculated to normalize for different fiber lengths, increases indicative of cell wall thickening were also observed beginning ∼22 DPA in both species. Subsequently, the fiber weight/length ratio increased similarly from 22 to 30 DPA in both species , the slopes of the linear regression lines (not shown) for fiber weight/length vs. DPA were 0.93 for Gh and 0.59 for Gb. This difference is consistent with the lower final fineness of Gb fiber (see below).Near 22 DPA in both species, the expression level of the secondary wall CESA genes reached their maximum level  and seco species . In contGb fiber walls observed in the transmission electron microscope (TEM) showed that adjacent fibers were joined together at 10, 17, and 19 DPA during early elongation, but the cell wall material between adjacent fibers (the CFML) had broken down by 24 DPA when the secondary wall had started to form ; Upper quartile length by weight, UQL(w); Length by number, L(n); and Longest 5% of fiber by number, L5%(n). The automated length measurement UQL(w) obtained by AFIS . Maturity ratio, a reflection of fiber wall thickness relative to fiber perimeter, was similarly high in the two species   compared to Gh . The greater fineness of Gb fiber is explained by its smaller perimeter and lower cellulose accumulation per unit length.As typically occurs under mutually suitable growing conditions, re cycle . All of  by AFIS  was simictively) . Use of Glycome Profiling Gh and Gb at 14 to 35 DPA are represented in a heat map  indicated the presence of these polymers in Gh fiber  XG in the 14 to 21 DPA Gh and Gb were joined together through 17 DPA when the CFML was still present, whereas they were separated at 24 DPA once the CFML had degraded -ß-Gal] sometimes found in XG Gh fiber Gb fiber was filled with HG (see the arrow in the panel showing 17 DPA fiber labeled with CCRC-M38), but the same bulge did not show any detectable signal with the other probes tested (see the white arrows pointing to the same location in other panels).The immunohistochemistry results were consistent with Glycome Profiling in showing the existence of several polymer classes within the primary cell walls that surround the protoplast in both species: XG (labeled with CCRC-M88 in the clade recognizing non-fucosylated XG); ß-1,3-glucan  (labeled with LAMP); HG (labeled with CCRC-M38); RG-I (labeled with CCRC-M35); and AG (labeled with CCRC-M107 and often existing as a side chain of RG-I). XG epitopes were detected in discrete spots corresponding to the bulges in the CFML only in Gh fiber had more XG, including fucosylated and non-fucosylated epitopes, in an unusual loosely-bound cell wall fraction as compared to Gb fiber. Hot acidic water is commonly used to straighten cotton fiber prior to hand measurement, and we predicted that this procedure would remove components of the CFML that helps cotton fiber to organize into curved bundles around each seed Gh fiber. Twelve antibodies were positive in dot blots of hot water extracts and in TEM immunolabeling of bulges in the CFML (unpublished data). These twelve antibodies recognized both fucosylated and non-fucosylated epitopes of XG and HG with no or relatively low esterification, corresponding to previous results A hot water extract of cotton fibers was used to confirm that Gh and Gb fiber at 10, 19, 24, and 30 DPA were probed in an immuno-dot assay with CCRC-M1 and CCRC-M58, which recognized fucosylated or non-fucosylated XG epitopes, respectively (Gh fiber (when the CFML was still present) had more XG than Gb fiber  corresponds to the continued detection of XG on the perimeter of Gh fiber at 24 and 35 DPA (The hot acidic water extracts of both ectively . Both prd 35 DPA .Gb fiber had up to four-fold higher XEH activity compared to Gh fiber (Xyloglucan endo-hydrolase (XEH) activity was measured in fiber protein extracts to look for evidence that the potential to degrade XG was different in the two species Gh fiber , then XEvia synthesis of nearly 100% cellulose  compared to the Gh fiber. Therefore, the moderate temperature and sufficient water and nutrients in the greenhouse supported normal fiber development even for the less adaptable Gb cotton plants. Despite their origins from the same (or similar) A and D ancestral genomes, these two allotetraploids have limited potential for interspecific hybridization, and the two species/cultivars we studied are distinct genotypes as indicated by a negligible calculated coefficient of parentage  similar rate of elongation before 22 DPA, when elongation ended in Gh; (b) the presence of a CFML; (c) a developmental shift in primary vs. secondary wall gene expression beginning ∼17 DPA; and (d) onset of secondary wall cellulose synthesis ∼22 DPA, along with the breakdown of the CFML. (See the data on the CFML of Gh fiber in Gb fiber (resulting in its greater length) and less cellulose deposited per unit length of Gb fiber (correlated at least in part with its lower diameter). In addition, the CFML composition was different as will be discussed further below. The highly resolved fiber growth profiles developed in parallel with harvesting fiber samples for analysis allowed strong correlation to be made between fiber cell wall chemistry and particular events in fiber development. These fiber growth profiles are likely to be useful to others when replicated with the same cultivars under the same growing conditions, but variance in any parameter may lead to the need to document the temporal progression of fiber development independently.Under the moderate and highly controlled greenhouse conditions used for our experiments, the fiber growth profiles in Gh fiber contain sugars typical of other primary walls: arabinose, fucose, galactose, glucose, mannose, rhamnose, xylose, glucuronic acid, and galacturonic acid, suggesting the presence of typical primary wall matrix polysaccharides Gh and Gb fiber cell walls by use of Glycome Profiling and immunohistochemistry, including XG, xylan, HG, RG-I, and AG (possibly as side chains of RG-I or AG proteins).The primary walls of Gh fiber with strong alkali had a ß-1,4-glucan backbone and typical XG oligosaccharide motifs G. arboreum, a diploid A genome cotton species Gh and/or Gb cotton fiber, although its appearance at the transition stage had been emphasized previously Gh fiber The results were consistent with a few studies where isolated cotton fiber wall polymers were chemically characterized. The XG purified from 19 DPA Gb fibers were joined by a CFML during the first 20 days of fiber development, as previously shown for Gh fiber Gb fiber continued to elongate rapidly after that time. Therefore, the CFML is not directly required for cotton fiber elongation to occur. It remains likely, however, that fiber bundle formation mediated by the CFML facilitates elongation indirectly through organizing >100,000 elongating fibers into efficiently packed tissue-like bundles within a confined space Adjacent Gb fiber is different compared to Gh fiber. Among the probes we tested, only the CCRC-M38 antibody that recognizes HG labeled the CFML bulges between Gb fibers, and HG was also found in the CFML of Gh fiber , but we cannot exclude the possibility that polymers or oligomers were present but were below the detection threshold. The 10 to 19 DPA Gb fiber had two- to four-fold higher XEH activity compared to Gh fiber, and the additional XEH activity could result in the formation of shorter XG fragments in Gb fiber. In future research, it will be worthwhile to isolate and compare the fine structure of XG, as well as potential related oligomers, in developing fiber of the two species. In transgenic G. hirsutum, higher capacity to transfer a XG chain end to a different XG polymer  that is rich in pectic arabinogalactans; a 1 M KOH extract prepared from switchgrass biomass (SG) that is rich in xylans; and a commercially available preparation of non-fucosylated tamarind xyloglucan (XG). Each polysaccharide preparation was assayed in water at concentrations from 1 to 20 µg/mL (A–C). Mixing experiments (D–F) were carried out by adding two different polysaccharide preparations in inverse concentrations to each other in order to end up with a total carbohydrate concentration of 20 µg/mL in each sample applied to the ELISA plates (50 µL volume). Antibodies used were: CCRC-M16, CCRC-M23, CCRC-M78 and CCRC-M133 recognize pectic arabinogalactan epitopes; CCRC-M150, CCRC-M153 and CCRC-M155 recognize xylan epitopes; and CCRC-M86, CCRC-M96, CCRC-M103 and CCRC-M111 recognize XG epitopes. The increase in the ELISA signal observed with increasing amounts of the AO extract (A) was unaffected by the presence of XG (D) or the SG extract (F). Likewise, the increase in ELISA signal observed with increasing amounts of the SG extract (B) was unaffected by the presence of XG (E) or AO extract (F). The increase in ELISA signal from tamarind XG (C) was similar in the presence of xylan in the SG extract (E). However, the presence of the pectic arabinogalactan-rich AO extract diminished the ELISA signal for the XG-directed antibodies tested at low concentrations of XG (up to 10 µg/mL). In contrast, at higher XG concentrations, the AO extract had little if any affect (D). These results suggest that at high pectic arabinogalactan concentrations, detection of low concentrations of XG could be compromised. However, the sequential cell wall extraction series, which typically separates most of the pectic arabinogalactans from the hemicelluloses, and the use of multiple antibodies against distinct epitopes within the same polymer class still allows Glycome Profiling to provide an accurate overall picture of cell wall composition.Effects of interaction between polysaccharides on ELISA absorbance values. Three different polysaccharide preparations were used in this experiment: an ammonium oxalate extract prepared from leaves of (DOC)Click here for additional data file.Figure S2Gh and Gb fiber samples shown in Overview (lower magnification) fluorescence micrographs of the (DOC)Click here for additional data file.Table S1MabDB plant cell wall mAbs database , which includes detailed descriptions of each mAb, including immunogen, antibody isotype, epitope structure recognized (where known), supplier information, and related literature references when available.Organization of antibodies into clades and links to additional information about the antibodies used in this study. The groupings of antibodies are based on a hierarchical clustering analysis of all monoclonal antibodies (mAbs) that were screened against a panel of plant polysaccharide preparations Click here for additional data file.Table S2ELISA absorbance values used to generate the heat map in (XLS)Click here for additional data file.


Initial steps in establishing an optimal strategy for functional bioengineered tissues is generation of three-dimensional constructs containing cells with the appropriate organization and phenotype. To effectively utilize rhesus monkey decellularized kidney scaffolds, these studies evaluated two key parameters: (1) residual scaffold components after decellularization including proteomics analysis, and (2) the use of undifferentiated human embryonic stem cells (hESCs) for recellularization in order to explore cellular differentiation in a tissue-specific manner. Sections of kidney and lung were selected for a comparative evaluation because of their similar pattern of organogenesis. Proteomics analysis revealed the presence of growth factors and antimicrobial proteins as well as stress proteins and complement components. Immunohistochemistry of recellularized kidney scaffolds showed the generation of Cytokeratin+ epithelial tubule phenotypes throughout the scaffold that demonstrated a statistically significant increase in expression of kidney-associated genes compared to baseline hESC gene expression. Recellularization of lung scaffolds showed that cells lined the alveolar spaces and demonstrated statistically significant upregulation of key lung-associated genes. However, overall expression of kidney and lung-associated markers was not statistically different when the kidney and lung recellularized scaffolds were compared. These results suggest that decellularized scaffolds have an intrinsic spatial ability to influence hESC differentiation by physically shaping cells into tissue-appropriate structures and phenotypes, and that additional approaches may be needed to ensure consistent recellularization throughout the matrix. For example, there is a need to explore fundamental questions related to scaffold properties including the contents that remain after the decellularization process and the tissue-specific properties that may potentiate lineage specification during recellularization. Differences in ECM composition, morphology, and structural properties are also known to impact scaffold physical properties and biological outcomes There is an ongoing interest in using native extracellular matrix (ECM) scaffolds derived from decellularized tissues as a potential method for repair of organs damaged by disease. Although results are promising, the clinical outcome of recent studies, such as those involving transplantation of tissue engineered decellularized airways, present issues such as the biomechanical collapse of the graft Studies by our group have demonstrated that rhesus monkey kidneys of all age groups  can be decellularized to provide a natural ECM with preserved structural and biological properties http://www.cnprc.ucdavis.edu). Euthanasia was consistent with the recommendations of the American Veterinary Medical Association (AVMA) Guidelines on Euthanasia and Primate Center SOPs . Tissue collection was performed using established methods and included juvenile (1–3 years) kidneys (transverse sections) (N = 3) and caudal lung lobes  (N = 3) from rhesus monkeys (Macaca mulatta) as previously described All animal protocols were approved prior to implementation by the Institutional Animal Care and Use Committee (IACUC) at the University of California, Davis, and all procedures conformed to the requirements of the Animal Welfare Act. Activities related to animal care including housing, feeding, and environmental enrichment were performed in accordance with IACUC-approved standard operating procedures (SOPs) at the California National Primate Research Center  on one decellularized kidney scaffold biopsy and one lung scaffold biopsy. The punch biopsy was collected from a transverse kidney section or sagittal caudal lung lobe section using a sterile 8 mm hollow Biopunch® . Samples were extracted by mechanical tissue disruption in a Bullet Blender  in 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% SDS, 0.05% ProteaseMax , 1X Complete Protease Inhibitor , and 1X PhoStop phosphatase inhibitor (Roche). Cell debris was pelleted via centrifugation and the supernatant was quantified using a Qubit fluorometer (Life Technologies). Each sample (20 µg) was loaded onto a 4–12% SDS-PAGE gel . The gel was excised into 40 segments per lane and gel slices were processed using a robot  for the following processing cycle: 25 mM ammonium bicarbonate wash, acetonitrile wash reduction with 10 mM dithiothreitol at 60°C, alkylation with 50 mM iodoacetamide at room temperature, digestion with trypsin (Promega) at 37°C for 4 h, and quenching with formic acid. The supernatant was analyzed directly without further processing.Each gel digest was analyzed by nano liquid chromatography and tandem mass spectrometry (LC/MS/MS) with a Waters NanoAcquity HPLC system interfaced to a ThermoFisher Orbitrap Velos Pro Peptides  and were loaded on a trapping column and eluted over a 75 µm analytical column at 350 nL/min; both columns were packed with Jupiter Proteo resin . The mass spectrometer was operated in data-dependent mode, with MS performed in the Orbitrap at 60,000 full width at half maximum (FWHM) resolution and MS/MS performed in the linear trap quadrupole (LTQ). The 15 most abundant ions were selected for MS/MS.M. mulatta (concatenated forward and reverse plus common contaminants); Database 2: NCBI M. mulatta (concatenated forward and reverse plus common contaminants); Fixed modification: Carbamidomethyl (C); Variable modifications: Oxidation (M), Acetyl (Protein N-term), Deamidation (NQ), Pyro-Glu (N-term Q); Mass values: Monoisotopic; Peptide Mass Tolerance: 10 ppm; Fragment Mass Tolerance: 0.8 Da; Max Missed Cleavages: 2.Data were searched using a local copy of Mascot with the following parameters: Enzyme: Trypsin; Database 1: Uniprot Mascot DAT files were parsed into Scaffold software  for validation, filtering, and to create a non-redundant list per sample. Data were filtered using a minimum protein value of 90%, a minimum peptide value of 50% (Prophet scores), and requiring at least two unique peptides per protein.M. mulatta and Uniprot M. mulatta databases. Peptides belonging to a given protein were normalized to the corresponding molecular weight. The abundance of each protein in the scaffold was assessed based on the percentage each occupied of the total normalized peptide spectra.Data were searched against both NCBI Prior to recellularization, scaffolds were washed overnight in sterile filtered  70% (v/v) ethanol in super-Q water at room temperature on a Dynal tube rotator  at 25 revolutions/min and rehydrated with several 24 h washes of PBS with 1% pen-strep.6 cells) to develop a standard curve to equate ε-globin (housekeeping gene) copy number with cell numbers. Juvenile kidney (N = 3) and lung (N = 3) scaffolds were cut into cylindrical biopsies (8 mm diameter) and seeded with approximately 5×105 undifferentiated hESCs on Day 0. Non-seeded decellularized kidney and lung scaffolds were also collected on Day 0 for histological analysis. Each seeded scaffold was suspended on PET track-etched membrane cell culture inserts . Once seeded, all scaffolds were cultured in Dulbecco's Modified Eagle's Medium high glucose with 10% fetal bovine serum (FBS), 1% pen-strep, and 1% L-glutamine at 37°C and 5% CO2. Two days after cells were seeded onto the scaffolds, each scaffold was gently transferred to new culture inserts to quantify non-adherent cells remaining in the original inserts with a hemacytometer and viability assessed with trypan blue. A reference Day 2 scaffold was collected from each group and stored at 4°C in 1 ml RNAlater® stabilization solution  for 48 h and then at ≤-80°C until processed for qPCR. On Day 8 scaffolds were collected for morphological analysis by fixing in 10% phosphate buffered formalin  and embedded in paraffin or collected for qPCR (stored at 4°C in 1 ml RNAlater® for 48 h and then at ≤-80°C until processed)  were cultured on irradiated mouse embryonic feeders and expanded from an established bank that was characterized as described previously for pluripotency markers and shown to spontaneously differentiate via embryoid bodies towards ectoderm, endoderm, and mesoderm ocessed) .5 hESCs were seeded onto Matrigel™  or were cultured in low adhesion plates to form embryoid bodies. Matrigel™ controls were allowed to form a semi-solid gel in the culture inserts to serve as three-dimensional (3D) ECM controls. Matrigel™ and embryoid body control samples (N = 3) were collected on day 8 of culture for immunohistochemistry (IHC) by dissolving Matrigel™ with cold PBS, followed by centrifugation at 1,500 revolutions per min (rpm) for 5 min at 4°C. Cell pellets for IHC were gently fixed in 10% formalin for 48 h and embedded in paraffin.Approximately 5×10hESCs were cultured in medium for embryoid body formation with solutions of 0%, 0.0001%, 0.001%, 0.01%, or 0.1% SDS (v/v) in low-adhesion plates for 8 days, with medium changes every 48 h. At the end of the culture period embryoid bodies were collected, washed with PBS, and centrifuged at 1,500 rpm for 5 min to form a pellet. Pellets were fixed in 10% formalin for 48 h and embedded in paraffin for IHC.To assess the effects of soluble molecules released from empty scaffolds on cell surfactant protein expression, empty decellularized juvenile kidney scaffolds (N = 3) were incubated with medium for 8 days. Every 24 h, the incubated medium was transferred from the empty scaffold culture to low adhesion plates containing hESCs for embryoid body formation. After 8 days, scaffolds and pellets were collected and fixed in 10% formalin for 48 h and embedded in paraffin for IHC.Morphology and scaffold recellularization were assessed by serial sectioning (5–6 µm) of the entire recellularized paraffin-embedded scaffold. Hematoxylin and Eosin (H&E) staining and IHC was performed as described 3,3′-Diaminobenzidine (DAB) staining was performed for HLA-E to assess MHC class I antigens in the decellularized scaffolds and for SP-B and SP-C to further assess surfactant expression in the recellularized kidney and lung scaffolds. Staining was performed using Dako EnVision+System-HRP (DAB) kit (Dako) according to the manufacturer’s instructions. Primary antibodies used were HLA-E  diluted 1∶100, SP-B diluted 1∶1000, and SP-C  diluted 1∶500 in primary antibody buffer with 5% goat serum. Slides were mounted with Cytoseal 60 mounting medium .2 Profiler™ PCR (Qiagen) array 96-well plates assessed gene expression of 11 kidney-associated genes or 11 lung-associated genes (later® solution at ≤-80°C using the AllPrep DNA/RNA Mini kit (Qiagen) following the manufacturer's instructions. Scaffolds were processed in multiple sections weighing <30 µg each to ensure efficient isolation of DNA and RNA.RT2 First Strand Kit (Qiagen) according to the manufacturer’s instructions. The RT2 RNA QC PCR array (Qiagen) was used to assess RNA quality prior to gene expression analysis. Real-time PCR reactions  were carried out in custom array plates with RT2 SYBR® Green ROX™ qPCR Mastermix (Qiagen) following instructions provided by the manufacturer using a 7900® ABI Sequence Detection System . The PCR protocol consisted of one cycle of 10 min at 95°C, followed by 40 cycles of 15 s at 95°C, and 1 min at 60°C. cDNA from three samples of undifferentiated hESCs were used as the calibrator.cDNA was synthesized using the RTCT method described in User Bulletin #2  relative to the housekeeping gene hypoxanthine phosphoribosyltransferase (HPRT) n-fold difference relative to a calibrator cDNA from three samples of undifferentiated hESCs.RNA expression was quantified according to the Comparative The number of cells seeded per scaffold was quantified by RT-PCR analysis of ε-globin after generation of a standard curve to equate copy number with cell number p ≤ 0.05 considered statistically significant.All data are shown as mean with the standard error of the mean (SEM). Statistical analysis was performed using a two tailed student’s t-Test with Our prior studies have shown intact cells are removed by the decellularization process as determined by the absence of H&E and DAPI staining of cell nuclei in decellularized kidney scaffolds Optimization of decellularization demonstrated that 1% SDS for kidney and 0.1% SDS for lung yielded scaffolds with preserved ECM structure while removing cells including MHC class I (HLA-E) and II (HLA-DR) antigens as assessed by IHC . AbsenceProteomics analysis identified 439 proteins in the kidney scaffold and 282 proteins in the lung scaffold (including known contaminants) with a false discovery rate of 0.2% for the kidney and 3.1% for the lung. After removal of known contaminants, there were 220 proteins detected in the kidney scaffold that were not present in the lung scaffold, 130 proteins expressed in the lung scaffold that were not present in the kidney scaffold, and 110 proteins expressed in both scaffolds. The top 100 most abundant proteins in each scaffold were grouped according to function . The mosThe scaffolds retained many proteins that have been viewed as potentially beneficial CT >39). These findings suggest that cellular mRNA, including common housekeeping genes, were effectively removed by the decellularization process and that residual gene expression in decellularized scaffolds would not interfere with qPCR results.Residual gene expression for kidney and lung-associated genes was assessed in decellularized kidney and lung scaffolds; however, the residual expression of the housekeeping gene, HPRT, was extremely low (Undifferentiated hESCs were seeded onto juvenile decellularized kidney or lung scaffolds. Gene expression of kidney or lung-associated genes was assessed by qPCR. Compared to baseline gene expression of undifferentiated hESCs (Day 0), expression levels of tubule markers, Dipeptidase 1 renal (DPEP1) and Heparan sulfate 6-O-sulfotransferase 1 (HS6T1), significantly increased after 2 days of culture on kidney scaffolds and remained elevated throughout the 8 days of culture. Other genes upregulated by culture on kidney scaffolds on Day 8 included fetal kidney tubule makers, Chloride Channel protein (CLCN7), and Homeobox protein B6 (HOXB6), as well as two markers of proximal tubules, Aminoacylase 1 (ACY1), and Fatty acid binding protein 1 (FABP1) . AlthougSeveral lung-associated genes including Bactericidal/permeability-increasing protein (BPI), which is expressed by respiratory epithelial cells of the bronchus and nasopharynx, SP-B, SP-C, and VWF, which are expressed by pneumocytes, were transiently elevated in kidney scaffolds by Day 2 but returned to baseline by Day 8 . SimilarExpression of kidney genes, UMOD  and Solute carrier family 12 (SLC12A), as well as the lung genes, Clara Cell specific protein  and TTF1/NK2 homeobox 1 (TTF1/NKX2.1), were not detected in cells cultured on either kidney or lung scaffolds. Compared to Day 0 undifferentiated hESCs, the cells cultured on kidney and lung scaffolds showed significant differences in tissue-specific gene expression over time, indicating that the scaffold was able to partially stimulate differentiation of hESCs to express tissue-specific markers. However, when gene expression levels of the differentiating cells on kidney versus lung scaffolds were compared on the same day, no statistically significant differences were found. These findings suggest that the gene expression response of hESCs to the scaffold ECM is similar for kidney and lung scaffolds despite statistical significance when compared to undifferentiated hESCs. Hence, the scaffolds were able to drive differentiation of hESCs to express kidney and lung genes, but no statistically significant difference in the gene expression patterns between hESCs seeded on kidney versus lung scaffolds were detected.Cells in the kidney scaffold formed thick symmetrical epithelial (Cytokeratin+) tubules, whereas cells in the lung scaffolds lined the alveolar spaces and formed thin networks of surfactant protein expressing cells . DespiteTo determine if the observed expression of surfactant proteins was confounded by residual staining of proteins remaining in the decellularized scaffold or was due to a response to a non-specific 3D ECM environment, IHC was performed on kidney and lung decellularized scaffolds (non-seeded) and hESCs cultured in Matrigel™, respectively. IHC performed on decellularized scaffolds prior to as well as following incubation with medium did not stain for surfactant proteins . These fTo examine the possible contribution of residual soluble molecules in the decellularized scaffolds on the stimulation of surfactant protein expression, embryoid bodies were exposed to various concentrations of SDS in culture medium or the supernatant from non-seeded decellularized scaffolds incubated in medium. Surfactant protein expression in embryoid bodies was not affected by the addition of SDS to the medium . StaininDecellularized tissue scaffolds provide a platform for developing functional tissue replacements by offering mechanical, structural, and biological properties similar to the native tissues from which they were derived. The first goal of the current study was to characterize the residual contents of kidney and lung scaffolds including DNA/RNA, MHC antigens, cellular markers, growth factors, and proteins to better understand and utilize the scaffolds for translational therapies. Kidney and lung were selected for a comparative evaluation because of their similar pattern of organogenesis, which is driven by reciprocal mesenchymal-to-epithelial interactions that lead to branching morphogenesis. Development of the kidney and lung depends on many of the same cytokines such as retinoids and bone morphogenic proteins It has been suggested that decellularized scaffolds retain cytokines that are beneficial for scaffold recellularization and host integration. Decellularized tracheal and brain matrices maintain expression of basic fibroblast growth factor-2 (FGF-2) and TGF-β, respectively. The presence of residual cytokines in decellularized matrices was sufficient to provide pro-angiogenic properties using the chicken embryo chorioallantoic membrane in vitro culture as well as downstream host integration. Potent antimicrobial and anti-fungal proteins such as Dermcidin-like protein found in kidney and lung scaffolds, and Defensin found in the lung, are consistent with findings by others who have demonstrated bacterial resistance of ECM-derived bladder and liver scaffolds In addition, decellularized tissues were shown to retain antimicrobial proteins that may be beneficial for These studies also highlight the importance of addressing the question of whether certain scaffold components and residual proteins may retain elements that could result in rejection post-transplantation. PCR revealed 96% DNA removal thus a small quantity of DNA remained emphasizing that additional steps such as enzymatic digestion, more vigorous mechanical agitation, or additional washes that affect pH or dielectric constants may be necessary to further enhance removal of residual DNA without damaging the ECM. Undigested DNA from dead cells and endogenous mitochondrial ’damage’-associated molecular patterns resulting from cellular trauma can cause activation of the innate immune response and elicit neutrophil-mediated organ injury creating a sepsis-like state in vivo environment, as well as additional decellularization strategies to enhance the removal of potentially immunogenic materials including more rigorous washing strategies to improve protein antigen removal Although proteomics analysis was performed on one kidney and one lung sample, additional samples would likely still reveal that ECM proteins only make up part of the several hundred proteins that remain in decellularized scaffolds. These findings suggest that in studies that utilize decellularized tissues, the cells are interacting with and responding to more than just the ECM. It is likely that the combination of ECM proteins with residual growth factors and other soluble molecules that remain in the decellularized scaffolds are responsible for directing differentiation and organization of the cells. This may be useful in cases of successful recellularization; however, these residual proteins may also be the cause of challenges encountered with consistent recellularization throughout the scaffold. It is not known if the successful use of decellularized tissues is only due to the structural benefits of the ECM proteins or if it is the combinatorial effects of ECM with an array of residual growth factors and molecules. However, preservation of these additional factors may also mean retention of other detrimental proteins that will present challenges for translation. Future studies will be needed to evaluate the potential consequences of the residual non-ECM proteins in an Several studies have suggested that residual SDS may prevent cell infiltration due to cytotoxicity. The protonated form of an amino group on lysine, which is displayed on ECM proteins, forms a strong bond to the negatively charged SDS A promising finding from this study is the ability of the scaffolds to physically shape tissue-appropriate structures. hESCs formed tubules in kidney scaffolds and the same hESC population differentiated into surfactant protein-secreting cells that organized around alveolar spaces in the lung scaffolds. The ECM composition and presentation of membrane-bound and soluble proteins differs for a renal tubule when compared to an alveolus; therefore, it is expected that different contact-dependent pathways may be stimulated in hESCs upon adhesion to different scaffolds that promote tissue-specific differentiation. Cells in both scaffolds expressed Cytokeratin, SP-B, and SP-C. Although additional analysis would need to be performed to clearly identify and characterize the differentiated cells within the scaffolds, Cytokeratin typically stains epithelial cells of the respiratory tract, including basal, ciliated, goblet, and alveolar cells To further characterize the differentiation state of the cells in each tissue scaffold, a panel of genes highly expressed in either kidney or lung was examined by qPCR. Here, results revealed that hESCs seeded onto decellularized kidney scaffolds acquired a significant increase in the expression of six kidney-associated markers whereas hESCs seeded onto lung scaffolds only acquired expression of one kidney-associated marker (ALPI). In contrast, short-term expression of lung-associated genes was observed regardless of the tissue scaffold on which undifferentiated hESCs were cultured. When examined in a longitudinal manner that followed gene expression of the cells over the entire culture period (8 days), statistically significant changes in tissue-specific gene expression were noted for cells on both scaffolds collected on Day 8 compared to the Day 0 undifferentiated hESC starting population. However, cross-comparison of a given gene for cells in kidney or lung scaffolds at a single time point demonstrated no statistical significance between the two scaffolds. These findings suggest that the scaffolds influence hESC differentiation but do not have the capability of genotypic lineage specification based solely on gene response. Alternative culture conditions may be required to induce appropriate and sustained tissue-specific gene expression. Because of their mechanistic similarities during ontogeny, the window of time included for the current study (8 days) may have limited the ability to fully capture divergence in gene expression of hESCs in kidney and lung scaffolds.To investigate the expression of surfactant proteins by cells in the recellularized kidneys, embryoid bodies were cultured in a range of SDS concentrations to assess the potential contribution of residual SDS on inducing expression of these proteins by cells. Although no correlation was found between increasing concentrations of SDS and surfactant protein expression, diffuse surfactant expression was noted for cells exposed to the culture supernatants of non-seeded decellularized kidney scaffolds. This finding suggests that a soluble factor in the empty decellularized scaffold is released into the medium during the culture period and is responsible for stimulating mild surfactant protein expression by embryoid bodies and likely the hESCs in the kidney and lung scaffolds as well. Additional proteomics analysis could be used to identify all soluble proteins that are released from the empty decellularized scaffolds while in culture. It is likely that one or more of these soluble molecules were responsible for the observed surfactant protein expression.In summary, this study highlights the potential use of decellularized tissues as a potent spatial mitogen and provides new insights into tissue-specific properties that can be exploited to drive cells from a pluripotent state to formation of tissue-specific structures without the addition of exogenous growth factors. Proteomics analysis of kidney and lung decellularized scaffolds also showed the inevitable translational hurdles in the form of hundreds of non-ECM residual proteins contained within scaffolds. Overall, these findings have demonstrated the ability of scaffold ECM to guide the spatial organization of cells into tissue-specific structures, although additional approaches will be needed to ensure consistent and efficient recellularization throughout the scaffold.Table S1Antibodies for Immunohistochemistry(PDF)Click here for additional data file.Table S2Kidney Genes for qPCR(PDF)Click here for additional data file.Table S3Lung Genes for qPCR(PDF)Click here for additional data file.Table S4Protein Composition of Kidney and Lung Scaffolds(PDF)Click here for additional data file.


In response to E. coli LPS, −/−Nlrp12 mice showed a slight decrease in IL-1β and increase in IL-6 production, but these levels were not statistically significant. During K. pneumoniae infection, we observed subtle differences in cytokine levels and significantly reduced numbers of monocytes and lymphocytes in −/−Nlrp12 mice. However, the physiological relevance of these findings is unclear as no overt differences in the development of lung disease were observed in the −/−Nlrp12 mice. Likewise, −/−Nlrp12 mice demonstrated pathologies similar to those observed in the wild type mice following M. tuberculosis infection. Together, these data suggest that NLRP12 does not significantly contribute to the in vivo host innate immune response to LPS stimulation, Klebsiella pneumonia infection or Mycobacterium tuberculosis.The majority of nucleotide binding domain leucine rich repeats-containing (NLR) family members has yet to be functionally characterized. Of the described NLRs, most are considered to be proinflammatory and facilitate IL-1β production. However, a newly defined sub-group of NLRs that function as negative regulators of inflammation have been identified based on their abilities to attenuate NF-κB signaling. NLRP12 (Monarch-1) is a prototypical member of this sub-group that negatively regulates both canonical and noncanonical NF-κB signaling in biochemical assays and in colitis and colon cancer models. The role of NLRP12 in infectious diseases has not been extensively studied. Here, we characterized the innate immune response of  Klebsiella pneumoniae and Mycobacterium tuberculosisInitiation and termination of the host innate immune response to pathogenic bacteria infection is tightly regulated. The host response to respiratory bacteria is initially driven through the stimulation of TLRs. TLR activation signals through the NF-κB pathway to up-regulate the transcription of proinflammatory cytokines, including IL-1β, TNFα, and IL-6, which are essential for the recruitment of leukocytes to the lung and clearance of the infection. Mice deficient in these three proinflammatory cytokines have demonstrated significant increases in morbidity and mortality during respiratory infections with While TLR recognition occurs on the plasma membrane and in endosomes, the nucleotide binding domain leucine rich repeats-containing family of proteins (NLRs) are a second family of pathogen recognition receptors (PRRs) that sense intracellular danger associated molecular patterns (DAMPs) and pathogen associated molecular patterns (PAMPs) in the cell cytoplasm In vivo, NLRP12 has been shown to dramatically influence the development of contact hypersensitivity, gastrointestinal inflammation and tumorigenesis Unlike the majority of characterized proinflammatory NLRs, recent studies have identified a novel sub-group of NLRs that have the ability to attenuate inflammation Klebsiella pneumoniae is a leading cause of community- and hospital-acquired respiratory infection. The growing prevalence of antibiotic resistant strains constitutes a serious public health concern K. pneumoniae is capable of inducing severe bacterial pneumonia that is characterized by extensive lung inflammation, hemorrhage and necrotic lesion formation in the lungs, which can often progress to bacteremia and sepsis K. pneumoniae, the incidence of Mycobacterium tuberculosis (Mtb) is also a global threat based on the increasing prevalence of antibiotic resistant MtbMtb and tuberculosis is associated with 1.4 million deaths per year Mtb develop a latent tuberculosis infection. Individuals that are latently infected with Mtb will maintain a significant lifetime risk of disease reactivation despite being asymptomatic Mtb infection is of immense clinical importance.Bacterial mediated infectious lung diseases are an important worldwide cause of morbidity and mortality. The gram negative bacterium K. pneumoniae and Mtb is influenced by NLR family members. Components of the NLRP3 inflammasome directly mediate the ex vivo and in vitro production of pro-inflammatory cytokines in response to K. pneumoniae infection in primary macrophages and monocytic cell lines K. pneumoniae in vivo through the regulation of IL-1β production and necrosis in the lungs Mtb. In addition, the NLR inflammasome adaptor protein PYCARD (ASC) exerts a novel NLRP3-independent role in granuloma formation and maintenance during in vivo Mtb infection Mtb in vitro. Studies in the THP-1 human monocytic cell line found that Nlrp12 is highly expressed at baseline levels, and expression is down-regulated following stimulation with TNF-α, IFN-γ, or MtbNLRP12 results in increased levels of pro-inflammatory cytokines following LPS or Mtb challenge in vitro with human cell lines that express high basal levels of NLRP12; thus, validation in a more physiologically relevant setting is necessary. While several studies have assessed the contribution of proinflammatory NLRs in host defense, few studies have explored the in vivo role of the NLR family members that exert negative regulatory activities in host defense against pathogenic bacteria.The innate immune response to in vivo characterization of proteins that function as negative regulators of inflammation is an area of intense scientific and clinical importance for the development of clinical therapies. Previous studies have shown that NLRP12 functions as a negative regulator of inflammatory pathways in vitro; however, the role of NLRP12 in regulating host-pathogen interactions in vivo is not well defined. Here, we tested the hypothesis that mice lacking Nlrp12 would demonstrate increased morbidity, mortality and pathology following in vivo K. pneumoniae or Mtb infection. Based on the previous in vitro findings, we expected to observe accelerated disease progression, lung inflammation and cytokine production associated with an overzealous immune response in the −/−Nlrp12 mice following K. pneumonia or Mtb bacterial infection.The identification and −/−Nlrp12 animals were kindly provided by Millennium Inc. and previously described −/−Nlrp12 mice were compared to both purchased C57Bl/6J mice and littermate control mice from heterozygous breeding. All in vivo Mtb work was conducted under approved BSL-3/ABSL-3 protocols and conditions.All studies were conducted under the approval of the Institutional Care and Use Committee (IACUC) for The University of North Carolina at Chapel Hill and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. −/−Nlrp12 dendritic cells were isolated and cultured as previously described E. coli 0111:B4 LPS (Invivogen), 50 ng/ml of Klebsiella pneumoniae LPS  or 50 µg/ml of trehalose-6,6-dibehenate (TDB), which is a synthetic analog of trehalose-6,6-dimycolate from Mycobacterium tuberculosis (Invivogen). Cell culture supernatants were collected following 4 hours of stimulation and TNF-α, IL-6 and IL-1β levels were assessed using ELISA (BD Biosciences). To evaluate IL-1β maturation and release, cells were treated with 2 mM ATP for 30 minutes prior to harvesting the supernatants. Pro- and cleaved IL-1β was detected by western blot in dendritic cell lysate and supernatant following 24 hours of LPS stimulation. Following the supernatant harvest, the remaining cells were lysed in 1% RIPA buffer. Immunoblots were probed with goat anti-mouse IL-1β primary antibody (R&D Biosystems), followed by rabbit anti-mouse HRP secondary antibody (Santa Cruz). Bands were visualized by Super Signal Chemiluminescence (Pierce).Wild type and Escherichia coli (sterile serotype 0111:B4) was instilled via intratracheal (i.t.) administration as previously described To assess lipopolysaccharide (LPS) induced acute airway inflammation, isoflurane inhalation was used to anesthetize the mice, and LPS (Sigma) isolated from K. pneumoniae lung challenge. K. pneumoniae 43816 (serotype 2) was obtained from the ATCC and cultured under suppliers recommended conditions. Prior to each experiment, bacteria density was estimated by measuring the absorbance at 600 nm and actual colony forming units (CFUs) were determined by plating an aliquot on LB agar plates. Mice were challenged via i.t. instillation with either 7.4×104 CFUs of K. pneumoniae in 50 µl of PBS or PBS alone (mock), as previously described in vivo bacteria burden, mice were euthanized via i.p. injection with 2,2,2 tribromoethanol (avertin), bronchoalveolar lavage fluid (BALF) was collected as described below, and the cell free fluid was serially diluted and plated on LB agar plates. Additionally, the liver was removed, wet weight assessed, homogenized in 500 µl of HBSS with a Tissue Master 125  and centrifuged. The resulting supernatants were plated on LB agar plates.Bacterial mediated acute airway inflammation was induced by Mycobacterium tuberculosis H37Rv strain was obtained from the American Type Culture Collection (ATCC). Bacteria were grown to log phase in Middlebrook 7H9 broth (Difco) with 0.2% glycerol, 1× albumin dextrose saline (ADS), and 0.05% Tween 80. Inoculum was assessed by plating infection media on Middlebrook 7H10 agar plates supplemented with glycerol and ADS. CFUs were counted after 21 days of incubation.in vivo airway infection, −/−Nlrp12 and wild type mice were infected via aerosol as previously described For K. pneumoniae models, mice were euthanized and serum was collected from animals by cardiac puncture. Bronchoalveolar lavage fluid (BALF) was collected to evaluate leukocytes and cytokine levels. The lungs were lavaged three times with 1 ml of HBSS. Following centrifugation, the cytokine levels in the cell free supernatants were evaluated by ELISA (BD Biosciences). Total BALF cellularity was determined from the resuspended cell pellet and evaluated using a hemocytometer. The composition of the BALF was determined by evaluating the morphology of ≥200 cells per sample following differential staining  of cells that were cytospun onto slides.Upon completion of the LPS and Mtb infection, mice were euthanized and the liver, spleen and right lung lobes were collected and homogenized for bacteria CFU analysis and cytokine profiles. Tissue homogenates were serial diluted and plated to determine CFUs. The remaining lung homogenates were centrifuged to remove cellular debris and the supernatant was double filter sterilized for cytokine analysis by ELISA.At specific time points post-K. pneumoniae histopathology, whole lungs were fixed by inflation to 20-cm pressure and immersion in 10% buffered formalin. To evaluate Mtb histopathology, the left lung lobe was removed, manually inflated and immersed in buffered formalin. Inflammation was evaluated in sections (5 µm) of the left lung lobe, following hematoxylin and eosin (H&E) staining. Paraffin embedded sections were set and cut to reveal the maximum longitudinal visualization of the intrapulmonary main axial airway. Histopathology was evaluated and scored by blinded reviewers on a scale of 0 (absent) to 3 (severe), as previously described Mtb histopathology as previously described For LPS and We utilized GraphPad Prism 5 Statistical software to conduct Analysis Of Variance (ANOVA) followed by either Tukey-Kramer HSD or Newman-Keuls post-test to evaluate statistical significance for multiple comparisons. Single data point comparisons were evaluated by the Student’s two-tailed t-test. All data are presented as the mean +/− the standard error of the mean (SEM), and in all cases a p-value of less than 0.05 was considered statistically significant.M. tuberculosisNlrp12 expression patterns in specific cell subpopulations. Nlrp12 expression in mouse cells associated with the host immune response was compiled using a publically accessible microarray meta-analysis search engine (Nextbio website. Available: http://www.nextbio.com/b/search/ba.nb. Accessed 2013 March 11), as previously described Nlrp12 in select cell populations relevant to the host immune response. Nlrp12 was found to be highly expressed in neutrophils and consistently expressed in dendritic cell populations  and the hyperphosphorylation of IRAK-1 −/−Nlrp12 and wild type mice were challenged with 50 µg of LPS through intratracheal (i.t.) administration and harvested 16–20 hrs post-administration. The LPS challenge resulted in a significant increase in total bronchoalveolar lavage fluid (BALF) cellularity, which was consistent with a significant increase in airway neutrophilia following LPS treatment  is a component of the cell wall of gram-negative bacteria and is ubiquitous in the environment. Inhalation of LPS has been shown to exacerbate airway reactivity in human asthmatics and activates the host innate immune system by acting as a PAMP to stimulate TLR4 receptors ex vivo data demonstrated that primary dendritic cells and neutrophils isolated from −/−Nlrp12 mice displayed significantly attenuated cell migration in a model of skin hypersensitivity −/−Nlrp12 and wild type mice  and systemic (serum) cytokine response following airway administration of LPS  is a chronic lung pathogen. Previous data obtained from an Mtb infected human macrophage line indicates that proinflammatory cytokine levels are increased in the absence of NLRP12 Mtb infection. Thus, we tested if the absence of NLRP12 would cause an overzealous host immune response leading to increased morbidity, mortality and lung inflammation during in vivo Mtb infection.In contrast to the acute lung infection caused by −/−Nlrp12 and wild type mice were infected with Mtb via aerosol infection. We found that the mean survival was 217 days post-infection for −/−Nlrp12 mice and 220 days for wild type mice . However, the physiological relevance of these findings is currently unclear and is in contrast to the anti-inflammatory role previously described for NLRP12 in human monocytic cell lines. This finding is reminiscent of NLRP3, which profoundly influences IL-1β production in cultured macrophages in response to Mtb, but does not significantly influence the in vivo model in vivo model of Mtb infection. Thus, we conclude that NLRP12 does not play a significant role in attenuating or exacerbating Mtb infection.To assess the host inflammatory response to Mtb infection results in a significant increase in lung inflammation and the formation of highly structured granulomas. A diverse array of cytokines has been shown to influence the formulation and maintenance of Mtb granulomas, including TNFα and IL-6 Mtb, but they are also important for regulating the host immune response during chronic Mtb infection. We observed a trending decrease in lung TNFα and IL-6 levels in the −/−Nlrp12 mice. Thus, we next sought to evaluate lung histopathology over the time course of Mtb infection. Lungs were harvested at 14, 26 and 181 days post-infection, which represents the early growth phase, the entry to the persistence phase and the chronic phase of Mtb infection in C57Bl/6 mice, respectively −/−Nlrp12 and wild type animals. In addition to assessments of inflammation, paraffin embedded lung sections were stained with Ziehl-Neelsen acid fast stain to identify Mtb in situ. Our earlier data suggested that the overall lung bacterial burden was unaffected by the loss of NLRP12 . However, it is possible that NLRP12 does contribute to the host innate immune response in a temporal, cell type-, tissue-, dose- or PAMP-specific manner and the models utilized here were too broad to discern its function. This hypothesis is supported by other findings that have shown differential and transient expression of NLRP12 in human and rodent cells in vivo with either silica particles or LPS in vivo bacteria models utilized in the current study are associated with robust immune responses, which may obscure the contribution of indirect or subtle regulators of innate immunity. Thus, it is possible that the in vivo contribution of NLRP12 may have been obscured by the high levels of inflammation, which was not a factor in the previous contact hypersensitivity study in vivo models, which combine lower levels of inflammation with higher resolution analyses, may reveal a role for NLRP12 in mediating the innate immune response to pathogens. For example, a recent study showed a role for NLRP12 in inflammasome formation and IL-1β/IL-18 maturation in response to a subcutaneous infection with a genetically attenuated strain of Yersinia, but not other activators that engage the NLRP3 inflammasome The most likely explanation for the discrepancy between the previous in vitro and in vivo findings is that other genes may be compensating for the loss of NLRP12 in vivo. Several recent papers have identified other NLRs that function as negative regulators of canonical and noncanonical NF-κB signaling, many of which may be capable of compensating for the loss of NLRP12. For example, NLRC5 has been shown to attenuate NF-κB signaling following in vitro PAMP stimulation A second possible explanation for the discordance between the in vivo assessments in the lung or in other tissues using other model systems may discern novel functions for NLRP12 in mediating innate immunity. Here, we assessed the role of NLRP12 in acute and chronic models of bacterial pneumonia and Mtb infection. Each of these diseases impact a growing number of humans world wide and it is essential that we expand our understanding of the underlying disease mechanisms associated with the host immune response in each of these cases. The lack of a role for NLRP12 in the disease processes assessed here is encouraging from the aspect of pharmacological inhibitors that target other inflammatory disorders, such as contact hypersensitivity or inflammatory bowel disease, where NLRP12 was found to play a role in a disease model.Our data suggests that NLRP12 does not contribute to host defense in response to a limited selection of PAMPs and pathogens. However, it should be noted that future


Deltaretroviruses such as human T-lymphotropic virus type 1 (HTLV-1) and bovine leukemia virus (BLV) induce a persistent infection that remains generally asymptomatic but can also lead to leukemia or lymphoma. These viruses replicate by infecting new lymphocytes (i.e. the infectious cycle) or via clonal expansion of the infected cells (mitotic cycle). The relative importance of these two cycles in viral replication varies during infection. The majority of infected clones are created early before the onset of an efficient immune response. Later on, the main replication route is mitotic expansion of pre-existing infected clones. Due to the paucity of available samples and for ethical reasons, only scarce data is available on early infection by HTLV-1. Therefore, we addressed this question in a comparative BLV model. We used high-throughput sequencing to map and quantify the insertion sites of the provirus in order to monitor the clonality of the BLV-infected cells population (i.e. the number of distinct clones and abundance of each clone). We found that BLV propagation shifts from cell neoinfection to clonal proliferation in about 2 months from inoculation. Initially, BLV proviral integration significantly favors transcribed regions of the genome. Negative selection then eliminates 97% of the clones detected at seroconversion and disfavors BLV-infected cells carrying a provirus located close to a promoter or a gene. Nevertheless, among the surviving proviruses, clone abundance positively correlates with proximity of the provirus to a transcribed region. Two opposite forces thus operate during primary infection and dictate the fate of long term clonal composition: (1) initial integration inside genes or promoters and (2) host negative selection disfavoring proviruses located next to transcribed regions. The result of this initial response will contribute to the proviral load set point value as clonal abundance will benefit from carrying a provirus in transcribed regions. Human T-lymphotropic Virus 1 (HTLV-1) induces a persistent infection that remains generally asymptomatic. Nevertheless, in a small proportion of individuals and after a long latency, HTLV-1 infection leads to leukemia or lymphoma. Onset of clinical manifestations correlates with a persistently elevated number of infected cells. Because the vast majority of cells are infected at early stages, primary infection is a crucial period for HTLV-1 persistence and pathogenesis. Since HTLV-1 is transmitted through breast feeding and because systematic population screenings are rare, there is a lack of available samples at early infection. Therefore, we addressed this question in a closely related animal model by inoculating cows with Bovine Leukemia Virus (BLV). We show that the vast majority of cells becoming infected during the first weeks of infection and do not survive later on. We also demonstrate that the initial host selection occurring during primary infection will specifically target cells that carry a provirus inserted in genomic transcribed regions. This conclusion thus highlights a key role exerted by the host immune system during primary infection and indicates that antiviral treatments would be optimal when introduced straight after infection. The deltaretrovirus genus includes human T-lymphotropic viruses (HTLVs), simian T-lymphotropic viruses (STLVs) and the bovine leukemia virus (BLV). These viruses induce a life-long persistent infection that remains generally asymptomatic  during primary infection. We demonstrated that (1) BLV initially targets transcribed regions of the genome for integration; (2) later, a massive clone selection occurs during primary infection disfavoring proviruses located nearby genes; (3) nevertheless, the abundance of the long term maintained clones benefits from the transcriptional activity of the genomic region surrounding the provirus.Five cows were inoculated with a cloned BLV provirus (strain 344) as described in the BLV inoculation into 5 cows thus resulted in the onset of an antibody response at days 16–30 which slightly preceded the maximal proviral burden (at days 30–68). This experimental setting is thus representative of BLV infection in herd conditions.To trace BLV clonality during primary infection, proviral insertion sites were selectively amplified, identified by high-throughput sequencing and quantified as schematized in Supporting Figure S1 in During primary infection, a broad range of clones were generated within each animal: from 264 in #21 to 12906 in #535  generally soon after seroconversion.The upper panel of BLV provirus integration did not occur randomly as illustrated on We conclude that BLV initial integration favors pol II and pol III transcribed regions of the genome.In contrast, BLV disfavors interspersed repeats such as LINE, SINE and ERVs . These eWe conclude that BLV initial integration disfavors transcriptionally silenced regions of the genome.We conclude that massive depletion of clones during primary infection is characterized by a preferential selection against proviruses inserted in transcribed regions.3 PBMCs) and was used as reference. The proportion of clones carrying a provirus located next to CpG islands or Refseq genes significantly increased with clone abundance . Although not significant, there was a similar trend for proximity with tRNA genes and tRNA pseudogenes. We conclude that the abundance of a long term established BLV-infected clone is enhanced by the integration of its provirus nearby a transcribed unit.Finally, we characterized the genomic environment of the proviruses that thrived in the long term. Therefore, we analyzed clonality in a series of cows infected for more than 2 years and harboring a wide range of PVL . These aHTLV-1 induces a persistent infection that is fortunately generally asymptomatic. Nevertheless, in a small proportion of individual and after a long latency, the infection leads to leukemia or lymphoma. These clinical manifestations are correlated with a persistently elevated proviral load and an oligo- or mono-clonal distribution of the infected cells. The same mechanism applies to BLV that induces leukemia or lymphoma in a minor fraction of infected animals. BLV also induces a persistent lymphocytosis in about 40% of the infected cows whereas this is far less common in HTLV-1 infected individuals. In fact, BLV and HTLV-1 are related deltaretroviruses sharing a similar genomic organization and infecting cells of the hematopoietic system (respectively mainly B cells and T cells). In this respect, BLV infection might be useful to address important questions unanswered in the HTLV-1 system. In particular, early infection appears to be critical at determining the different populations of cells (i.e. clones) that will subsequently thrive and expand during pathogenesis We first show that thousands of clones are generated during primary infection indicating that initial replication occurs through the infectious cycle. None of the newly generated clones massively expanded during this early period revealing a polyclonal pattern of viral expansion and confirming observations made in the BLV-infected sheep model Therefore, the BLV model can be informative to understand mechanisms of early infection by deltaretroviruses, a period that cannot be easily addressed in the HTLV-1 system as discussed previously due to the logistic and ethical problems of collecting sequential neonatal samples. In BLV-inoculated sheep, the time period delimiting the infectious cycle was estimated to last the first 8 months Currently, there are no available data pertaining to the early viral replication in HTLV-1 infected patients. Based on this study and previous publications using animal models A major contribution of our report is to demonstrate the massive depletion of the early generated clones. In It has been previously shown that HTLV-1 infected clones are not equal regarding their proliferative potential. Indeed, the abundance of a given clone is enhanced by the integration of its provirus in an actively transcribed area of the genome At a first glance, the observation that clone abundance positively correlates with provirus proximity with transcribed units appears to conflict with the extremely low levels of viral expression measured in vivo In summary, we have characterized BLV clonality during primary infection using high throughput sequencing of the proviral insertion sites in 7 sequential samples of 5 BLV-inoculated cows and as well as in long term infected animals. We demonstrate that BLV proviruses initially integrate into transcribed regions of the genome but are massively depleted later on. This mechanism may have important outcomes for HTLV-1 prevention and treatment.Cows were experimentally infected with the wild-type BLV strain 344. Two 15 cm-diameter dishes containing subconfluent Hela cells were transfected with 80 µg of plasmid pBLV344 containing the BLV provirus, recovered in 5 ml PBS at day 3 and injected subcutaneously. The presence of anti-BLV antibodies was determined using a competitive ELISA test (IDEXX Leukosis Blocking Ab Test). Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll Hypaque density gradient centrifugation (Sigma-Aldrich), washed and cryopreserved in fetal calf serum with 10% DMSO (Sigma-Aldrich).Animal experimentations were conducted in accordance with national and international guidelines for animal care and use described in the Manual for use and care of experimental animals emitted by INTA. Handling of cows and experimental procedures were reviewed and approved by INTA's Institutional Committee for Care and Use of Experimental Animals (CICUAE-INTA) under protocol number 35/2010.5′-GAAACTCCAGAGCAATGGCATAA-3′ and 5′-GGTTCGGCCATCGAGACA-3′. As reference for genomic DNA, β-actin was amplified with oligonucleotides 5′-TCCCTGGAGAAGAGCTACGA-3′ and 5′-GGCAGACTTAGCCTCCAGTG-3′. Three dilutions of DNA  were amplified by real-time quantitative PCR in a Roche light cycler using MESA green master mix (Eurogentec). The thermal protocol used started with a 95°C 5 min denaturation step; then 45 cycles as follows  and terminated with a melting curve. PCR efficacies were calculated for each sample using the three dilutions. Standard curves were generated using PCR4topo vectors (Life Technologies) containing the corresponding pol or actin amplicon. Proviral load was calculated, as an average of the three dilutions, from the number of proviral copies divided by half of the number of actin copies and expressed as number of proviral copies per 100 of PBMCs.DNA was extracted from PBMCs using DNeasy Blood and Tissue kit (Qiagen). BLV DNA was PCR amplified using pol gene sequence-specific primers Diagenode Bioruptor instrument using the following protocol . DNA ends were then end-repaired using 15 units of T4 DNA polymerase (New England Biolabs), 5 units of DNA polymerase I Klenow fragment (NEB), 50 units of T4 polynucleotide kinase (NEB) and 0.8 mM of dNTP (Sigma) in T4 DNA ligase buffer (NEB) at 20°C during 30 min. DNA was then cleaned using a Qiaquick PCR purification kit (Qiagen) and eluted in 64 µl of EB buffer. Addition of an adenosine at the 3′ ends of the DNA was performed by adding 0.2 mM of dATP (Sigma) and 15 units of Klenow Fragment 3′ to 5′ exo- (NEB) in NEB2 buffer (NEB) at 37°C for 30 min. DNA was then cleaned using a Qiaquick PCR purification kit and eluted in 40 µl of EB. One hundred pmol of a partially double stranded DNA linker was ligated to the DNA ends using a Quick ligation kit (NEB). Twenty four different linkers were designed, each one with a specific 8 bp tag (see Primer list in Ten micrograms of genomic DNA extracted from PBMCs were sheared by sonication with the i (number of cells per 100 PBMCs) was calculated from the number of sister cells and the measurement of the proviral load as follows:iX is the number of sister cells of the ith clone, D the number of observed clones and PVL the proviral load.“Sister cells” are cells where the BLV provirus is inserted at the same site in the cellular genome and a “clone” is a population of sister cells. For each unique insertion site, the number of amplicons of different length (i.e. different shear sites) give an estimate of the number of sister cells of that infected clone i  was expressed as follows:The relative abundance of a given clone To measure the clonality of the infected cell population, i.e. the non-uniformity of the clone abundance distribution, we used the oligoclonality index based on the Gini coefficient vice versa). Because this index takes clone abundance into account, populations that contain the same clones but have different clone abundance will have an index value of less than 1. Similarity indexes were calculated between 2 successive time points separated by two weeks. Details of the calculations are given in supplemental file To measure the likeliness or overlap between two populations of BLV-infected cells, we calculated a similarity index. This number ranges from 0 to 1, with 0 indicating that no clones are shared between the two populations and 1 corresponding to a complete identity  when p>0.05 and NA for Non Applicable.Text S1The supporting file (PDF)Click here for additional data file.