Increased autophagy in EphrinB2-deficient osteocytes is associated with elevated secondary mineralization and brittle bone

Mineralized bone forms when collagen-containing osteoid accrues mineral crystals. This is initiated rapidly (primary mineralization), and continues slowly (secondary mineralization) until bone is remodeled. The interconnected osteocyte network within the bone matrix differentiates from bone-forming osteoblasts; although osteoblast differentiation requires EphrinB2, osteocytes retain its expression. Here we report brittle bones in mice with osteocyte-targeted EphrinB2 deletion. This is not caused by low bone mass, but by defective bone material. While osteoid mineralization is initiated at normal rate, mineral accrual is accelerated, indicating that EphrinB2 in osteocytes limits mineral accumulation. No known regulators of mineralization are modified in the brittle cortical bone but a cluster of autophagy-associated genes are dysregulated. EphrinB2-deficient osteocytes displayed more autophagosomes in vivo and in vitro, and EphrinB2-Fc treatment suppresses autophagy in a RhoA-ROCK dependent manner. We conclude that secondary mineralization involves EphrinB2-RhoA-limited autophagy in osteocytes, and disruption leads to a bone fragility independent of bone mass.Mineralized bone forms when collagen-containing osteoid accrues mineral crystals. This is initiated rapidly (primary mineralization), and continues slowly (secondary mineralization) until bone is remodeled. The interconnected osteocyte network within the bone matrix differentiates from bone-forming osteoblasts; although osteoblast differentiation requires EphrinB2, osteocytes retain its expression. Here we report brittle bones in mice with osteocyte-targeted EphrinB2 deletion. This is not caused by low bone mass, but by defective bone material. While osteoid mineralization is initiated at normal rate, mineral accrual is accelerated, indicating that EphrinB2 in osteocytes limits mineral accumulation. No known regulators of mineralization are modified in the brittle cortical bone but a cluster of autophagy-associated genes are dysregulated. EphrinB2-deficient osteocytes displayed more autophagosomes in vivo and in vitro, and EphrinB2-Fc treatment suppresses autophagy in a RhoA-ROCK dependent manner. We conclude that secondary mineralization involves EphrinB2-RhoA-limited autophagy in osteocytes, and disruption leads to a bone fragility independent of bone mass

Transcriptional Profiling of Chondrodysplasia Growth Plate Cartilage Reveals Adaptive ER-Stress Networks That Allow Survival but Disrupt Hypertrophy

Metaphyseal chondrodysplasia, Schmid type (MCDS) is characterized by mild short stature and growth plate hypertrophic zone expansion, and caused by collagen X mutations. We recently demonstrated the central importance of ER stress in the pathology of MCDS by recapitulating the disease phenotype by expressing misfolding forms of collagen X (Schmid) or thyroglobulin (Cog) in the hypertrophic zone. Here we characterize the Schmid and Cog ER stress signaling networks by transcriptional profiling of microdissected mutant and wildtype hypertrophic zones. Both models displayed similar unfolded protein responses (UPRs), involving activation of canonical ER stress sensors and upregulation of their downstream targets, including molecular chaperones, foldases, and ER-associated degradation machinery. Also upregulated were the emerging UPR regulators Wfs1 and Syvn1, recently identified UPR components including Armet and Creld2, and genes not previously implicated in ER stress such as Steap1 and Fgf21. Despite upregulation of the Chop/Cebpb pathway, apoptosis was not increased in mutant hypertrophic zones. Ultrastructural analysis of mutant growth plates revealed ER stress and disrupted chondrocyte maturation throughout mutant hypertrophic zones. This disruption was defined by profiling the expression of wildtype growth plate zone gene signatures in the mutant hypertrophic zones. Hypertrophic zone gene upregulation and proliferative zone gene downregulation were both inhibited in Schmid hypertrophic zones, resulting in the persistence of a proliferative chondrocyte-like expression profile in ER-stressed Schmid chondrocytes. Our findings provide a transcriptional map of two chondrocyte UPR gene networks in vivo, and define the consequences of UPR activation for the adaptation, differentiation, and survival of chondrocytes experiencing ER stress during hypertrophy. Thus they provide important insights into ER stress signaling and its impact on cartilage pathophysiology

Phagocytic capacity of leucocytes in sheep mammary secretions following weaning

Lactating animals are particularly susceptible to mastitis during the early stages of mammary gland involution following weaning. In this study we compared the phagocytic capacity of cells collected from sheep mammary secretions at different stages of involution. The ability of neutrophils and macrophages to ingest latex beads in an in vitro phagocytosis assay was found to be dependent on how heavily the phagocytes were loaded with milk constituents. There was a decline in the phagocytic capacity of neutrophils from 1 to 2 days after weaning, while macrophages collected from fully involuted glands were more effective phagocytes compared with earlier stages (7–15 days) of involution. In addition, dendritic cells present in fully involuted mammary gland secretions (30 days after weaning) were highly phagocytic. These studies demonstrate that neutrophils and macrophages in sheep mammary secretions at early stages of involution are incapacitated, and as such may compromise the immune status of the mammary gland

Univariable analysis assessing select epidemiological variables as predictors for the presence of herpesvirus DNA in eastern grey kangaroos ^a.

<p>^a Reference levels are indicated by odds ratio of 1.0. Results highlighted in bold (log likelihood p ≤ 0.25) represent variables included in the initial multivariable model, with the exception of presence of pouch young/lactation as it is correlated with sex and thus excluded. Backward elimination of non-significant variables yielded no significant variables. Multivariable analysis was repeated including presence of pouch young/lactation as a variable instead of sex. Age was excluded from the model due to collinearity. In the final model (n = 42) only the absence of pouch young/lactation was identified as a significant factor (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133807#pone.0133807.t008" target="_blank">Table 8</a>). n/a = not applicable.</p><p>Univariable analysis assessing select epidemiological variables as predictors for the presence of herpesvirus DNA in eastern grey kangaroos <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133807#t004fn001" target="_blank">^a</a>.</p

Univariable analysis assessing select epidemiological variables as predictors for the presence of active herpesvirus infection in common wombats ^a.

<p>^a Reference levels are indicated by odds ratio of 1.0. Results highlighted in bold (log likelihood p ≤0.25) represent variables included in the initial multivariable model, with the exception of pouch young which was excluded due to zero prevalence of herpesvirus infection in females lactating/with pouch young. In the final model (n = 33) only age (adult/aged) and body condition score (≤ 2) were identified as significant factors (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133807#pone.0133807.t008" target="_blank">Table 8</a>). n/a = not applicable.</p><p>Univariable analysis assessing select epidemiological variables as predictors for the presence of active herpesvirus infection in common wombats <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133807#t007fn001" target="_blank">^a</a>.</p

Electron micrographs of novel herpesviruses.

<p>Transmission electron microscopy was used to visualise herpesviruses in cultures of primary wombat kidney cells. Herpesvirus capsids (arrowheads) of VoHV-1 (A) and VoHV-2 (B) are shown. Bar = 100 nm.</p

Anatomical sites of herpesvirus DNA detection in swab samples collected from Australian marsupials in 2010 and 2011.

<p>^a Only species that were sampled in relatively large numbers, from multiple anatomical sites, are included.</p><p>^b Herpesvirus DNA was sometimes detected in more than one swab from the same animal, swabs were not collected from every anatomical site from every animal.</p><p>Anatomical sites of herpesvirus DNA detection in swab samples collected from Australian marsupials in 2010 and 2011.</p

Univariable analysis assessing select epidemiological variables as predictors for the presence of herpesvirus DNA in Tasmanian devils ^a.

<p>^a Reference levels are indicated by odds ratio of 1.0. Results highlighted in bold (log likelihood p ≤ 0.25) represent variables included in the initial multivariable model, with the exception of season as it was directly influenced by timing of management procedures, and therefore correlated with captive status and was thus excluded. In the final model (n = 50) only captivity was identified as a significant factor (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133807#pone.0133807.t008" target="_blank">Table 8</a>). n/a = not applicable.</p><p>Univariable analysis assessing select epidemiological variables as predictors for the presence of herpesvirus DNA in Tasmanian devils <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133807#t006fn001" target="_blank">^a</a>.</p

Univariable analysis assessing select epidemiological variables as predictors for the presence of herpesvirus DNA in koalas ^a.

<p>^a Reference levels are indicated by odds ratio of 1.0. Results highlighted in bold (log likelihood p ≤ 0.25) represent variables included in the initial multivariable model, with the exception of season as the timing of sampling correlated with the location at which it occurred and was thus excluded. In the final model (n = 68) only the presence of <i>Chlamydia pecorum</i> was identified as a significant factor (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133807#pone.0133807.t008" target="_blank">Table 8</a>). n/a = not applicable.</p><p>Univariable analysis assessing select epidemiological variables as predictors for the presence of herpesvirus DNA in koalas <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133807#t005fn001" target="_blank">^a</a>.</p

Predicted amino acid alignment and phylogenetic tree of the novel marsupial herpesviruses.

<p>A: Alignment of the predicted amino acid sequence of a portion of the DNA polymerase gene of the novel marsupial herpesviruses, along with other herpesviruses from the three herpesvirus sub-families. B: Maximum likelihood tree generated from the alignment. Bootstrap values of 100 replicates are displayed on the tree branches. Novel herpesvirus species are underlined. Key: PaHV-2 = papiine herpesvirus 2 (AAN87165.1); SaHV-1 = saimiriine herpesvirus 1 (YP_003933809.1); HHV-1 = human herpesvirus 1 (NP_044632.1); MaHV-1 = macropodid herpesvirus 1 ([<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133807#pone.0133807.ref022" target="_blank">22</a>]); <u>VoHV-3 = vombatid herpesvirus 3 (novel sequence)</u>; PCMV = porcine cytomegalovirus (AF268042.1); HHV-6 = human herpesvirus 6A (NP_042931.1); HHV-4 = human herpesvirus 4 (YP_401712.1); HHV-8 = human herpesvirus 8 (ACY00400.1); SaHV-2 = saimiriine herpesvirus 2 (NP_040211.1); BHV-4 = bovine herpesvirus 4 (NP_076501.1); DaHV-1 = dasyurid herpesvirus 1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133807#pone.0133807.ref013" target="_blank">13</a>]; <u>DaHV-2 = dasyurid herpesvirus 2</u> (novel sequence); MaHV-3 = macropodid herpesvirus 3 (ABO61861.1); <u>MaHV-5 = macropodid herpesvirus 5 (novel sequence). PeHV-1 = peramelid herpesvirus 1 (novel sequence</u>); PotHV-1 = potoroid herpesvirus 1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133807#pone.0133807.ref010" target="_blank">10</a>]; <u>VoHV-1 = vombatid herpesvirus 1 (novel sequence)</u>; <u>VoHV-2 = vombatid herpesvirus 2 (novel sequence);</u> PhaHV-1 = phascolarctid herpesvirus 1 (AEX15649.1); PhaHV-2 = phascolarctid herpesvirus 2 (AFN66528.1); EHV-2 = equine herpesvirus 2 (NP_042605.1); CpHV-2 = caprine herpesvirus 2 (ADV92276.1).</p