Regulation of Feto-Maternal Barrier by Matriptase- and PAR-2-Mediated Signaling Is Required for Placental Morphogenesis and Mouse Embryonic Survival

<div><p>The development of eutherian mammalian embryos is critically dependent on the selective bi-directional transport of molecules across the placenta. Here, we uncover two independent and partially redundant protease signaling pathways that include the membrane-anchored serine proteases, matriptase and prostasin, and the G protein-coupled receptor PAR-2 that mediate the establishment of a functional feto-maternal barrier. Mice with a combined matriptase and PAR-2 deficiency do not survive to term and the survival of matriptase-deficient mice heterozygous for PAR-2 is severely diminished. Embryos with the combined loss of PAR-2 and matriptase or PAR-2 and the matriptase partner protease, prostasin, uniformly die on or before embryonic day 14.5. Despite the extensive co-localization of matriptase, prostasin, and PAR-2 in embryonic epithelia, the overall macroscopic and histological analysis of the double-deficient embryos did not reveal any obvious developmental abnormalities. In agreement with this, the conditional deletion of matriptase from the embryo proper did not affect the prenatal development or survival of PAR-2-deficient mice, indicating that the critical redundant functions of matriptase/prostasin and PAR-2 are limited to extraembryonic tissues. Indeed, placentas of the double-deficient animals showed decreased vascularization, and the ability of placental epithelium to establish a functional feto-maternal barrier was severely diminished. Interestingly, molecular analysis suggested that the barrier defect was associated with a selective deficiency in the expression of the tight junction protein, claudin-1. Our results reveal unexpected complementary roles of matriptase-prostasin- and PAR-2-dependent proteolytic signaling in the establishment of placental epithelial barrier function and overall embryonic survival.</p></div

A combined loss of PAR-2 and matriptase leads to underdevelopment of placental labyrinth.

<p>(<b>A, B</b>). Immunohistochemical detection of matriptase (<b>A</b>) and prostasin (<b>B</b>) in mouse placenta at E12.5. Expression of both proteins was observed in chorionic epithelium (<b>A</b> and <b>B</b>, arrowheads) and in the differentiated syncytiothrophoblast layer of the labyrinth (<b>A</b> and <b>B</b>, arrows). Prostasin, but not matriptase, was also detected in the mononuclear cytotrophoblast cells within the labyrinth (<b>B</b>, open arrowheads). (<b>C</b>). Histological analysis of PAR-2 expression in the developing placenta from E12.5 <i>F2rl1-βgal</i> knock-in embryos. Beta-galactosidase reporter activity was detected both in chorionic epithelium (arrowheads) and in the syncytiothrophoblast layer surrounding fetal vessels within the labyrinth (arrows). (<b>D, E</b>). Histological evaluation of the placental development in control (<b>D</b>) and <i>F2rl1^−/−;St14^−/−</i> (<b>E</b>) animals at E13.5. H&E staining confirmed the presence of all major structural components of the mouse placenta, including allantoic mesenchyme (al), placental labyrinth (lb), spongiotrophoblast layer (sp, examples with open arrowheads), and trophoblast giant cells (tgc, examples with arrowheads), in the double-deficient embryos (<b>F–I</b>). Immunohistochemical visualization of fetal vasculature in the E13.5 placentas. Low (<b>F, H</b>) and high (<b>G, I</b>) magnification images of the staining for the endothelial cell marker PECAM-1/CD31 in the placentas of the control (<b>F, G</b>) and <i>F2rl1^−/−;St14^−/−</i> (<b>H,I</b>) mice shows presence of the branched fetal vascular tree (<b>F–I</b>, arrows) within the placental labyrinth of the PAR-2/matriptase double-deficient animals, although the apparent vascular density is lower than in the wildtype littermate control tissues. (<b>J, K</b>). Quantification of the thickness of the placental labyrinth (<b>J</b>) and the number of fetal vessels (<b>K</b>) within the labyrinth of control (<i>F2rl1⁺;St14⁺</i>) and <i>F2rl1^−/−;St14^−/−</i> E12.5 and E13.5 placentas. The measurements show decreased thickness and vascularization of the labyrinth layer in <i>F2rl1^−/−;St14^−/−</i> placentas at E12.5 and E13.5. P values: *<0.05, **<0.01, ***<0.001, Student's t-test, two-tailed. Scale bars: (<b>A, B</b>) 50 µm, (<b>C</b>) 100 um, (<b>D, E, F, H</b>) 200 µm, (<b>G, I</b>) 50 µm.</p

Loss of PAR-2 and matriptase does not affect development of the embryo proper.

<p>(<b>A–C</b>). Evaluation of the expression pattern of <i>St14</i> (<b>A</b>), <i>Prss8</i> (<b>B</b>), and <i>F2rl1</i> (<b>C</b>) genes in E14.5 mouse embryo by <i>in situ</i> hybridization obtained from the Eurexpress digital transcriptome atlas. All three genes show highly overlapping pattern of expression in the epithelia of developing skin (sk), oral (oc) and nasal (nc) cavities, salivary gland (sg), lungs (l), kidney (k), and gut (g). Matriptase and prostasin, but not PAR-2, were also detected in the developing structures of the inner ear (ie). (<b>D</b>). Macroscopic appearance of <i>F2rl1^−/−;St14^−/−</i>, and wild-type littermate control embryos at E13.5. No obvious developmental abnormality was observed in the PAR-2 and matriptase double-deficient animals. (<b>E</b>) Total body weight of E13.5 offspring from <i>F2rl1^+/−;St14^+/−</i> breeding pairs. None of the embryos with a decreased combined gene dosage of <i>F2rl1</i> and <i>St14</i> genes showed any signs of growth retardation. (<b>G–I′</b>). H&E staining of embryonic tissues with the highest relative expression of PAR-2 and matriptase. A comparative histological analysis of liver (<b>F, F′</b>), lungs (<b>G, G′</b>), kidneys (<b>H, H′</b>), and intestines (<b>I, I′</b>) from the control (<b>F, G</b>, <b>H</b>, and <b>I</b>) and littermate <i>F2rl1^−/−;St14^−/−</i> (<b>F′, G′</b>, <b>H′</b>, and <b>I′</b>) E13.5 embryos indicates normal development of embryonic tissues in the absence of PAR-2 and matriptase function. Scale bars: (<b>A–C</b>) 1 mm, (<b>F–I′</b>) 100 um.</p

Combined loss of matriptase/prostasin- and PAR-2-dependent proteolytic pathways leads to embryonic lethality.

<p>(<b>A</b>). Matriptase haploinsufficiency decreases survival of PAR-2-deficient mice. Genotype distribution among 706 pre-weaning offspring from interbred <i>F2rl1^+/−</i>×<i>F2rl1</i>^+/−<i>;St14^+/−</i> mice. A normal distribution of <i>F2rl1</i> alleles was observed in <i>St14^+/+</i> background, whereas the number of <i>F2rl1^−/−</i> mice heterozygous for matriptase was significantly decreased (P<0.0001). (<b>B</b>). Genotype distribution among 272 newborn offspring from interbred <i>F2rl1^+/−;St14^+/−</i>×<i>F2rl1</i>^+/−;<i>St14^+/−</i> mice. <i>F2rl1</i> alleles were found in the expected Mendelian ratio in <i>St14^+/+</i> mice, whereas numbers of <i>F2rl1^−/−;St14^+/−</i> and <i>F2rl1^+/−;St14^−/−</i> mice was significantly reduced, and no <i>F2rl1^−/−;St14^−/−</i> mice were observed (P<0.0001). (<b>C</b>). Relative prenatal survival of PAR-2-deficient (<i>F2rl1^−/−</i>, blue bars) and matriptase-deficient (<i>St14^−/−</i>, green bars) mice shown in (<b>B</b>) as a function of gene dosage of <i>St14</i> and <i>F2rl1</i> genes, respectively. Survival of PAR-2-deficient animals decreased from 90% in <i>St14^+/+</i> background, to 40% in <i>St14^+/−</i> and 0% in <i>St14^−/−</i> background. Similarly, survival of matriptase-deficient mice decreased from 73% in <i>F2rl1^+/+</i> animals to 35% in <i>F2rl1^+/−</i> and 0% in the PAR-2-deficient <i>F2rl1^−/−</i> animals. (<b>D</b>). Genotype distribution among 247 newborn offspring from interbred <i>F2rl1^+/−;Prss8^+/−</i>×<i>F2rl1</i>^+/−;<i>Prss8^+/−</i> mice. <i>F2rl1</i> alleles were found in the expected Mendelian ratio in <i>Prss8^+/+</i> mice, whereas numbers of <i>F2rl1^−/−;Prss8^+/−</i> and <i>F2rl1^+/−;Prss8^−/−</i> mice was significantly reduced, and no <i>F2rl1^−/−;Prss8^−/−</i> mice were observed (P<0.0005). (<b>E, F</b>). Survival of <i>F2rl1^−/−;St14^−/−</i> (<b>E</b>) and <i>F2rl1^−/−;Prss8^−/−</i> (<b>F</b>) embryos before birth, relative to the expected Mendelian distribution. (<b>E</b>). PAR-2/matriptase double-deficient embryos were detected in expected numbers at or before E12.5, followed by a decrease to 59% at E13.5 (N = 251), and 15% at E14.5 (N = 90). No surviving <i>F2rl1^−/−;St14^−/−</i> animals were detected at or after E15.5. (<b>F</b>). PAR-2 and prostasin double-deficient embryos were detected in expected numbers at E11.5, followed by a gradual decrease to 80% at E12.5 (N = 90), 47% at E13.5 (N = 176), and 17% at E14.5 (N = 68). No surviving <i>F2rl1^−/−;Prss8^−/−</i> animals were detected at or after E15.5.</p

Placental expression of matriptase restores survival in PAR-2 and matriptase double-deficient embryos.

<p>(<b>A</b>). Schematic depiction of the strategy used to assess contribution of placental matriptase to the survival of the <i>F2rl1^−/−;St14^−/−</i> embryos. <i>Meox2-Cre</i> allele that drives the expression of Cre recombinase to the embryonic but not placental tissues was introduced into the mice carrying one null and one conditional allele of matriptase gene (<i>St14^−/fl</i>). The resulting mice retain expression of matriptase in the placenta but not in any of the embryonic tissues. Both the embryo and the placenta are also PAR-2-deficient (<i>F2rl1^−/−</i>). (<b>B</b>). Western blot analysis of matriptase expression in the embryo (top) and the placenta (bottom) of the E13.5 <i>Meox2-Cre;St14^−/fl</i> (lanes 1–3), and their <i>St14^+/fl</i> (lanes 4–6) and <i>St14^−/−</i> (lane 7) littermate control animals. Both the embryos and the placentas of the control mice showed detectable levels of matriptase protein (arrowheads on the right), whereas only placental tissue retained matriptase expression in the <i>Meox2-Cre;St14^−/fl</i> embryos. No matriptase expression was detected in either embryos or placentas of animals carrying two knockout alleles of matriptase (<i>St14^−/−</i>). GAPDH signal is shown to indicate equal loading. Positions of molecular weight markers (kDa) are shown on left. (<b>C</b>). Allele distribution of the <i>F2rl1</i> gene among newborn matriptase-deficient offspring from interbred <i>F2rl1^+/−;St14^+/−</i>×<i>F2rl1</i>^+/−;<i>St14^+/−</i> (<i>St14^−/−</i>, left panels) or <i>Meox2-Cre;F2rl1^+/−;St14^+/−</i>×<i>F2rl1^+/−;St14^−/fl</i> (<i>Meox2-Cre;St14^−/fl</i>, right panels) breeder mice. Placental expression of matriptase restores embryonic survival of the PAR-2 and matriptase double-deficient mice. (<b>D, E</b>). Macroscopic appearance of the head of control (<b>D</b>) or <i>Meox2-Cre;F2rl1^−/−;St14^−/fl</i> (<b>E</b>) mice at birth. The <i>Meox2-Cre;F2rl1^−/−;St14^−/fl</i> newborns reproduce phenotypes of mice with complete matriptase deficiency, including the lack of whiskers. (<b>F</b>). Western blot analysis of matriptase expression in two <i>Meox2-Cre;F2rl1^−/−;St14^−/fl</i> (lanes 1–2) and two littermate control (lanes 3–4) newborn mice. No residual expression of matriptase protein (arrowhead on the right) was detected in the <i>Meox2-Cre;F2rl1^−/−;St14^−/fl</i> mice. Positions of molecular weight markers (kDa) are shown on left.</p

Loss of matriptase and PAR-2 function impairs formation of the feto-maternal barrier.

<p>(<b>A</b>). Relative uptake of the transcellular transport marker 3-methyl-D-glucose injected into the maternal bloodstream by E12.5–13.5 embryos. Placentas of matriptase (<i>F2rl1^+/+,St14^−/−</i>) or PAR-2 (<i>F2rl1^−/−;St14^+/+</i>) single-deficient embryos supported glucose transport at a rate comparable to the wildtype littermate controls (<i>F2rl1⁺;St14⁺</i>), whereas matriptase and PAR-2 double-deficient mice (<i>F2rl1^−/−;St14^−/−</i>) showed about 20% decrease in the glucose uptake (P<0.01). (<b>B</b>). Relative uptake of the paracellular transport marker carboxy-inulin injected into the maternal bloodstream by E12.5–13.5 embryos. Diffusion of carboxy-inulin across the placental epithelium was strongly dependent on gene dosage of both matriptase and PAR-2, showing 247% and 145%, respectively, increase in <i>St14^−/−</i> and <i>F2rl1^−/−</i> single-deficient embryos, and 402% and 351% increase, respectively, in <i>F2rl1^+/−;St14^−/−</i> and <i>F2rl1^−/−;St14^+/−</i> embryos, compared to the wildtype littermate controls. Highest levels of inulin transfer across the placenta, 589% above the control, was observed in mice with a combined matriptase and PAR-2 deficiencies (<i>F2rl1^−/−;St14^−/−</i>). (<b>C</b>). Expression of placental labyrinth differentiation markers syncytin A (upper panel) and GCMa (lower panel) in the placental tissues from E12.5 matriptase- and PAR-2-expressing (control, lanes 1–3) or matriptase- and PAR-2 double-deficient (<i>F2rl1^−/−;St14^−/−</i>, lanes 4–6) embryos. No change in the expression of either of the two proteins was detected. (<b>D, E</b>). Western blot analysis (<b>D</b>) and protein signal quantification (<b>E</b>) of the major structural components of epithelial tight junctions (claudin-1 and -2), adherens junctions (pan-cadherin), and desmosomes (desmoglein-1 and -2), or GAPDH as the protein loading control in the placental tissues from E12.5 matriptase- and PAR-2-expressing (control, lanes 1–4) or matriptase and PAR-2 double-deficient (<i>F2rl1^−/−;St14^−/−</i>, lanes 5–8) embryos. No significant differences were observed in the expression of claudin-2, cadherins, or desmoglein 1 and 2, whereas the expression of claudin-1 was significantly reduced. P values: *<0.05, ** <0.01, *** <0.001, N.S. = not significant, Student's t-test, two-tailed.</p

Data_Sheet_1_Intrathecal B Cells in MS Have Significantly Greater Lymphangiogenic Potential Compared to B Cells Derived From Non-MS Subjects.XLSX

<p>Although B cell depletion is an effective therapy of multiple sclerosis (MS), the pathogenic functions of B cells in MS remain incompletely understood. We asked whether cerebrospinal fluid (CSF) B cells in MS secrete different cytokines than control-subject B cells and whether cytokine secretion affects MS phenotype. We blindly studied CSF B cells after their immortalization by Epstein-Barr Virus (EBV) in prospectively-collected MS patients and control subjects with other inflammatory-(OIND) or non-inflammatory neurological diseases (NIND) and healthy volunteers (HV). The pilot cohort (n = 80) was analyzed using intracellular cytokine staining (n = 101 B cell lines [BCL] derived from 35 out of 80 subjects). We validated differences in cytokine production in newly-generated CSF BCL (n = 207 BCL derived from subsequent 112 prospectively-recruited subjects representing validation cohort), using ELISA enhanced by objective, flow-cytometry-based B cell counting. After unblinding the pilot cohort, the immortalization efficiency was almost 5 times higher in MS patients compared to controls (p < 0.001). MS subjects' BCLs produced significantly more vascular endothelial growth factor (VEGF) compared to control BCLs. Progressive MS patients BCLs produced significantly more tumor necrosis factor (TNF)-α and lymphotoxin (LT)-α than BCL from relapsing-remitting MS (RRMS) patients. In the validation cohort, we observed lower secretion of IL-1β in RRMS patients, compared to all other diagnostic categories. The validation cohort validated enhanced VEGF-C production by BCL from RRMS patients and higher TNF-α and LT-α secretion by BCL from progressive MS. No significant differences among diagnostic categories were observed in secretion of IL-6 or GM-CSF. However, B cell secretion of IL-1β, TNF-α, and GM-CSF correlated significantly with the rate of accumulation of disability measured by MS disease severity scale (MS-DSS). Finally, all three cytokines with increased secretion in different stages of MS (i.e., VEGF-C, TNF-α, and LT-α) enhance lymphangiogenesis, suggesting that intrathecal B cells directly facilitate the formation of tertiary lymphoid follicles, thus compartmentalizing inflammation to the central nervous system.</p

Matriptase Deletion Initiates a Sjögren’s Syndrome-Like Disease in Mice

<div><p>Objective</p><p>The objective of this study was to determine the effect of epithelial barrier disruption, caused by deficiency of the membrane-anchored serine protease, matriptase, on salivary gland function and the induction of autoimmunity in an animal model.</p><p>Methods</p><p>Embryonic and acute ablation of matriptase expression in the salivary glands of mice was induced, leading to decreased epithelial barrier function. Mice were characterized for secretory epithelial function and the induction of autoimmunity including salivary and lacrimal gland dysfunction, lymphocytic infiltration, serum anti-Ro/SSA, anti-La/SSB and antinuclear antibodies. Salivary glands immune activation/regulation, barrier function as well as tight junction proteins expression also were determined. Expression of matriptase in minor salivary gland biopsies was compared among pSS patients and healthy volunteers.</p><p>Results</p><p>Embryonic ablation of matriptase expression in mice resulted in the loss of secretory epithelial cell function and the induction of autoimmunity similar to that observed in primary Sjögren’s syndrome. Phenotypic changes included exocrine gland dysfunction, lymphocytic infiltrates, production of Sjögren’s syndrome-specific autoantibodies, and overall activation of the immune system. Acute ablation of matriptase expression resulted in significant salivary gland dysfunction in the absence of overt immune activation. Analysis of the salivary glands indicates a loss of electrical potential across the epithelial layer as well as altered distribution of a tight junction protein. Moreover, a significant decrease in matriptase gene expression was detected in the minor salivary glands of pSS patients compared with healthy volunteers.</p><p>Conclusions</p><p>Our findings demonstrate that local impairment of epithelial barrier function can lead to loss of exocrine gland dysfunction in the absence of inflammation while systemic deletion can induce a primary Sjögren’s syndrome like phenotype with autoimmunity and loss of gland function.</p></div

T cell activation and regulation changes in draining lymph nodes and spleens from <i>St14</i>^– mice.

<p>Salivary gland draining lymph nodes (DLN) and spleens were collected from <i>St14⁺</i> and <i>St14^–</i> animals. Samples from 2–3 mice were pooled per group and used to detect CD4, CD8 and CD62L expression by flow cytometry. (A, B) Both CD4+ and CD8+ cells were significantly decreased in both the DLN and spleens from <i>St14^–</i> mice compared with <i>St14⁺</i> mice. CD62L expression was also decreased on both CD4+ and CD8+ cells in <i>St14^–</i> mice compared with <i>St14⁺</i> mice. (C, D, E) The percentage and total number of CD4+CD25+Foxp3+ natural T regulatory cells (nTreg) were altered in the DLN and spleen cells. Data shown are the mean ± SEM for each animal group (<i>N</i> = 4 for both groups). Statistical significance was determined using an unpaired Student’s <i>t</i>-test. *<i>P</i><0.05, **<i>P</i>≤0.001, ***<i>P</i><0.0001.</p

Decreased epithelial electrical potential as well as changes in protein distribution in mice with acute salivary gland-specific ablation of matriptase.

<p>Electrical potential (EP) of salivary glands was measured 22 weeks post-AAV2 delivery to glands in live AAV2-Cre (<i>N = </i>5) and AAV2-LacZ (<i>N = </i>3) mice. Membrane potential of pierced ducts of <i>St14^LoxP/LoxP</i> mice (<i>N = </i>2) was used as a control for impaired ductal epithelium. (A) Local depletion of matriptase in SMG from AAV2-Cre animals resulted in a significant EP decrease compared with SMG from AAV2-LacZ control mice. The EP of AAV2-Cre mice was similar to that of pierced glands. (B) Correlation between saliva production and EP of salivary glands from AAV2-Cre mice (<i>N = </i>3) and AAV2-LacZ mice (<i>N = </i>3). Data shown are the mean ± SEM for each group. Statistical significance was determined using unpaired Student’s <i>t</i> test and Pearson’s rank correlation test, respectively. NS, <i>P = </i>0.1978, **<i>P = </i>0.0044, ***<i>P</i><0.001. (C) Changes in distribution of proteins associated with tight junctions or salivary gland function induced by the loss of matriptase were visualized by immunofluorescent staining of paraffin-embedded SMG tissue samples from <i>St14^–</i> and <i>St14⁺</i> control mice 28 to 40 weeks of age (<i>N = </i>2 both groups). Representative confocal images are shown. Apical staining of ductal cells for claudin 3 is shown by arrowheads in both <i>St14⁺</i> and <i>St14⁻</i> animals. An increase in the signal for claudin 3 was detected in the cytoplasm and basal membrane of ductal cells in <i>St14^–</i> mice compared with <i>St14⁺</i> mice, as indicated with arrow; original magnification 40X.</p