Single-Cell Analysis Reveals Isotype-Specific Autoreactive B Cell Repertoires in Sjögren’s Syndrome

Microengraving is a novel technology that uses an array of microfabricated subnanoliter wells to isolate and characterize secreted proteins from larger number of single cells. This printing technique permits the capture and characterization of secreted antibodies on glass slides. Here, we profiled the antigenic repertoires of B cells reacting against salivary gland tissues in Sjögren’s syndrome (SjS), an autoimmune disease targeting the exocrine glands. Single-cell suspensions of spleen and cervical lymph node cells prepared from normal C57BL/6 and SjS-susceptible (SjSs) C57BL/6.NOD-AecAec2 mice were dispersed into subnanoliter wells (nanowells). Capture slides preincubated with mouse immunoglobulins were used for printing. Detection antibodies included fluorescence conjugated anti-IgG1, salivary gland lysates of C57BL/6 and SjSs mice. Results indicate an increase in the frequency of IgG1-secreting cells in the spleen of SjSs mice compared to C57BL/6 mice. Cells from the lymph node of SjSs mice yield higher instances of IgG1 reactive against salivary gland antigens than cells from the lymph nodes of C57BL/6 mice. These data demonstrate the isotype-specific reactivity of antibodies during the autoimmune process, and further reveals significant differences in the non-autoimmune and autoimmune antibody repertoires. These results support the generation of self-reactive B cell repertoires during the autoimmune process, at the same time, verifying that microengraving of single cells might allow for identification of novel biomarkers in SjS.Sjogren's Syndrome FoundationNational Institute of Dental and Craniofacial Research (U.S.) (PHS Grant R00 DE018958)National Institutes of Health (U.S.) (NIH/NIAID (RC1AI086152)

Illustration of microengraving.

<p>Arrays of nanowells with dimensions of 50 µm×50 µm×50 µm were used for microengraving. Spleen or cervical lymph nodes cells were loaded in the nanowells. Cells in the nanowells were imaged using an automated epifluorescence microscope. Micrograving is performed by hybridizing nanowells with capture slides containing anti-mouse Ig for 2 hrs at 37°C with 5% CO₂. After incubation, nanowells containing intact live cells and capture slides were separated. A mixture of antibodies containing IgG1-Alexa Fluor 488, B6 SG lysate-Alexa Fluor 594 and <i>Aec1Aec2</i> SG lysate-Alexa Fluor 555 were added to the capture slides. Micrographs of microarrays were generating by scanning using a Genepix 4200AL microarray scanner.</p

Characterization of cells from the cervical lymph nodes of C57BL/6 and C57BL/6.NOD-<i>Aec1Aec2</i> mice using microengraving.

<p><b>A.</b> Representative micrographs of live cells from C57BL/6 cervical lymph nodes (n = 4) in nanowells labeled with Calcein (live cells), CD19-FITC and CD4-Cy7. Micrographs of matching microarray showing detection signals for IgG1-Alexa Fluor 488, C57BL/6 (B6) salivary glands proteins labeled with Alexa Fluor 594 and C57BL/6.NOD-<i>Aec1Aec2</i> (SjS) salivary glands proteins labeled with Alexa Fluor 555. The last vertical panel illustrates the close-up features (arrows) (Live cell: CD19FITC, IgG1: IgG1-488 signal, B6 gland: signal of antibody binding to salivary gland proteins isolated from B6 mice. SjS gland: signal of antibody binding to salivary proteins isolated from SjS mice. <b>B.</b> Representative micrographs of live cells from C57BL/6.NOD-<i>Aec1Aec2</i> cervical lymph nodes (n = 4) in nanowells labeled with Calcein (live cells), CD19-FITC and CD4-Cy7. Micrographs of matching microarray showing detection signals for IgG1-Alexa Fluor 488, B6 salivary glands proteins labeled with Alexa Fluor 594 and SjS salivary glands proteins labeled with Alexa Fluor 555. The last vertical panel illustrates the close-up features pointed by the arrows (Live cell: CD19FITC, IgG1: IgG1-488 signal, B6 gland: signal of antibody binding to salivary gland proteins isolated from B6 mice. SjS gland: signal of antibody binding to salivary proteins isolated from SjS mice. All experiments were repeated at least twice for consistency.</p

Frequency of IgG1.

<p>Enumeration of IgG1-secretion cells from arrays of nanowells occupied by single cells from the spleens and cervical lymph nodes of C57BL/6 (n = 4) and C57BL/6.NOD-<i>Aec1Aec2</i> mice (n = 4). Data extracted from the image processing using Genepix software were used to identify the appropriate signals. The data were correlated with the nanowell image data in which nanowells contained a single cell positive for both Calcein (live cells) and CD19. The frequency was determined by using the ratio of positive IgG1 signal from wells with single cells and the total number of wells with single cells. *p<0.05 by unpaired t test. NS: not significant.</p

Zinc Transporter ZIP14 Functions in Hepatic Zinc, Iron and Glucose Homeostasis during the Innate Immune Response (Endotoxemia)

<div><p>ZIP14 (slc39A14) is a zinc transporter induced in response to pro-inflammatory stimuli. ZIP14 induction accompanies the reduction in serum zinc (hypozincemia) of acute inflammation. ZIP14 can transport Zn²⁺ and non-transferrin-bound Fe²⁺ in vitro. Using a <em>Zip14^−/−</em> mouse model we demonstrated that ZIP14 was essential for control of phosphatase PTP1B activity and phosphorylation of c-Met during liver regeneration. In the current studies, a global screening of ZIP transporter gene expression in response to LPS-induced endotoxemia was conducted. Following LPS, Zip14 was the most highly up-regulated Zip transcript in liver, but also in white adipose tissue and muscle. Using <em>ZIP14^−/−</em> mice we show that ZIP14 contributes to zinc absorption from the gastrointestinal tract directly or indirectly as zinc absorption was decreased in the KOs. In contrast, <em>Zip14^−/−</em> mice absorbed more iron. The <em>Zip14</em> KO mice did not exhibit hypozincemia following LPS, but do have hypoferremia. Livers of <em>Zip14−/−</em> mice had increased transcript abundance for hepcidin, divalent metal transporter-1, ferritin and transferrin receptor-1 and greater accumulation of iron. The <em>Zip14^−/−</em> phenotype included greater body fat, hypoglycemia and higher insulin levels, as well as increased liver glucose and greater phosphorylation of the insulin receptor and increased GLUT2, SREBP-1c and FASN expression. The <em>Zip14</em> KO mice exhibited decreased circulating IL-6 with increased hepatic SOCS-3 following LPS, suggesting SOCS-3 inhibited insulin signaling which produced the hypoglycemia in this genotype. The results are consistent with ZIP14 ablation yielding abnormal labile zinc pools which lead to increased SOCS-3 production through G-coupled receptor activation and increased cAMP production as well as signaled by increased pSTAT3 via the IL-6 receptor, which inhibits IRS 1/2 phosphorylation. Our data show the role of ZIP14 in the hepatocyte is multi-functional since zinc and iron trafficking are altered in the <em>Zip14^−/−</em> mice and their phenotype shows defects in glucose homeostasis.</p> </div

Deletion of <i>Zip14</i> in mice produces altered glucose homeostasis and IR functions.

<p>(A) Body composition of the WT and Zip14^−/− female mice was measured using a NMR Lean/Fat analyzer. (B) Serum and liver glucose from fed-mice were measured by OneTouch UltraMini and colorimetrically, respectively. (C, D) Serum insulin and liver cAMP were measured by ELISA. (E, F) Western analysis results from liver of three mice are shown for each treatment group. (G) Total RNA was isolated from livers and relative transcript abundance for GLUT2, PEPCK, SREBP-1c, FASN and SOCS-3 were measured by qPCR and expressed relative to TBP mRNA as the normalizer. Values are mean ± SE, n = 3−5.</p

LPS differentially regulates ZIP14 expression in mice.

<p>Young adult mice received LPS (2 mg/kg, i.p.) or the same volume (0.5 mL) of saline (control), 1–18 hr before being killed. (A) Total RNA was isolated and <i>Zip14</i> mRNA was measured by qPCR and expressed relative to TBP mRNA as the normalizer. ZIP14 protein abundance was measured by western analysis of liver homogenates. Representative western blots from multiple mice (n = 3−4) were measured for ZIP14 abundance by densitometry. (B, C) Zinc concentrations in serum and liver, in µg/mL and µg/g respectively, were measured by AAS. (D, E) Comparison of Zip14 mRNA and ZIP14 protein in WT and <i>Zip14</i> KO mice 18 hr after LPS, as measured by qPCR and western analysis. Values are mean ± SD, n = 3−5. (E) ZIP14 protein is increased at the plasma membrane of hepatocytes of WT mice but not Zip14^−/− mice following LPS. Localization was by confocal microscopy using ZIP14 antibody and Alexa fluor594 secondary antibody and DAPI as the nuclear marker. (F) Serum IL-6 as measured by ELISA was used as an indicator of efficiency of LPS administration. The IL6 response from LPS was attenuated in the <i>Zip14^−/−</i> mice. (* = P<0.05, ** = P<0.01, *** = P<0.001, **** = P<0.0001).</p

<i>Zip14^−/−</i> mice exhibit normal iron absorption but altered iron homeostasis.

<p>(A) WT and Zip14^−/− mice were administered LPS (2 mg/kg or saline 0.5 mL; ip), 18 hr before being killed. (A) Serum and liver iron concentrations were measured by AAS. Liver non-heme-iron was measured colorimetrically. (B) Fasted WT and <i>Zip14^−/−</i> mice received 2 µCi of ⁵⁹Fe by gavage and were killed 24 hr later. Percent absorption was calculated from the radioactivity administered. Serum and liver iron uptake was calculated from the specific activity of the ⁵⁹Fe. (C) Transcript abundance for liver hepcidin, TfR-1, DMT1 and ferritin was measured by qPCR and expressed relative to TBP mRNA as the normalizer. Values are mean ± SE, n = 5−10.</p

<i>Zip14</i> null mice do not have depressed serum zinc after LPS administration.

<p>WT and <i>Zip14^−/−</i> mice were given LPS or saline administration (2 mg/kg; 0.5 mL; i.p.) 18 hr before being killed. (A, B) Zn concentrations in serum and liver were measured by AAS. (C, D) Global analysis of liver Zip and ZnT transcripts in KO and after LPS. Total RNA was isolated from the liver and <i>Zip14</i> mRNA was measured by qPCR and normalized to TBP mRNA. Values are mean ± SD, n = 3−5. (E) ZIP4 protein abundance in liver was shown by western analysis.</p

Single-Cell Analysis Reveals Isotype-Specific Autoreactive B Cell Repertoires in Sjögren’s Syndrome

Microengraving is a novel technology that uses an array of microfabricated subnanoliter wells to isolate and characterize secreted proteins from larger number of single cells. This printing technique permits the capture and characterization of secreted antibodies on glass slides. Here, we profiled the antigenic repertoires of B cells reacting against salivary gland tissues in Sjögren’s syndrome (SjS), an autoimmune disease targeting the exocrine glands. Single-cell suspensions of spleen and cervical lymph node cells prepared from normal C57BL/6 and SjS-susceptible (SjSs) C57BL/6.NOD-AecAec2 mice were dispersed into subnanoliter wells (nanowells). Capture slides preincubated with mouse immunoglobulins were used for printing. Detection antibodies included fluorescence conjugated anti-IgG1, salivary gland lysates of C57BL/6 and SjSs mice. Results indicate an increase in the frequency of IgG1-secreting cells in the spleen of SjSs mice compared to C57BL/6 mice. Cells from the lymph node of SjSs mice yield higher instances of IgG1 reactive against salivary gland antigens than cells from the lymph nodes of C57BL/6 mice. These data demonstrate the isotype-specific reactivity of antibodies during the autoimmune process, and further reveals significant differences in the non-autoimmune and autoimmune antibody repertoires. These results support the generation of self-reactive B cell repertoires during the autoimmune process, at the same time, verifying that microengraving of single cells might allow for identification of novel biomarkers in SjS.Sjogren's Syndrome FoundationNational Institute of Dental and Craniofacial Research (U.S.) (PHS Grant R00 DE018958)National Institutes of Health (U.S.) (NIH/NIAID (RC1AI086152)