Inducible nitric oxide synthase-mediated injury in a mouse model of acute salivary gland dysfunction.

AIM: Inducible nitric oxide synthase (iNOS) is a key regulator of the innate immune system. The aim of the current study was to explore whether innate immune-mediated iNOS and reactive nitrogen species acutely perturb acinar cell physiology and calcium homeostasis of exocrine salivary tissues. METHODS: Innate immunity in the submandibular gland of C57BL/6 mice was locally activated via intraductal retrograde infusion of polyinosinic:polycytidylic acid (poly (I:C). Expressions of iNOS and the activity of the reactive nitrogen species peroxynitrite, were evaluated by immunohistochemistry. Mice were pre-treated with the selective iNOS inhibitor aminoguanidine in order to substantiate the injurious effect of the nitrosative signal on the key calcium regulator sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2b) and calcium signalling. RESULTS: Challenging salivary gland innate immunity with poly (I:C) prompted upregulated expression of iNOS and the generation of peroxynitrite. Inhibition of iNOS/peroxynitrite revealed the role played by upregulated nitrosative signalling in: dysregulated expression of SERCA2b, perturbed calcium homeostasis and loss of saliva secretion. CONCLUSION: iNOS mediates disruption of exocrine calcium signalling causing secretory dysfunction following activation of innate immunity in a novel salivary gland injury model.

Epithelial disruptions, but not immune cell invasion, induced secretory dysfunction following innate immune activation in a novel model of acute salivary gland injury.

BACKGROUND: Salivary gland (SG) injurious agents are all translated into loss of salivation (xerostomia). An association has been established between activation of innate immunity and SG injury and dysfunction. However, it remains unclear how the secretory epithelia respond by halting saliva production. METHODS: C57BL/6 submandibular glands (SMGs) were acutely challenged using a single dose of the innate immune stimulant: polyinosinic-polycytidylic acid (poly (I:C)). Secretory capacity of the infected SMGs was substantiated by assessing the flow rate in response to pilocarpine stimulation. Depletion of the acute inflammatory cells was achieved by pre-treating mice with RB6-8C5 depletion antibody. Flow cytometry, histology and immunohistochemistry were conducted to verify the immune cell depletion. Epithelial expression of saliva-driving molecules: muscarinic 3 receptor (M3R), aquaporin 5 water channel (AQP5), Na-K-CL-Cotransporter 1 (NKCC1) and transmembrane member 16A (TMEM16A), was characterized using RT-qPCR and immunohistochemistry. Tight junction (TJ) protein; zonula occludens (ZO-1) and basement membrane (BM) protein; and laminin were assessed by immunohistochemistry. RESULTS: Innate immune challenge prompted dysfunction in the exocrine SGs. Dysregulated gene and protein expression of molecules that drive saliva secretion was substantiated. Aberrant expression of TJ and BM proteins followed innate immune activation. Hyposalivation in the current model was independent of myeloperoxidase (MPO)-positive, acute inflammatory cells. CONCLUSIONS: In this study, we developed a novel injury model of the SGs, featuring acute secretory dysfunction and immediate structural disruptions. Our results ruled out the injurious role of aggressively infiltrating inflammatory cells.

Correction: Ruchaya et al. Transplantation of Skeletal Muscle-Derived Sca-1+/PW1+/Pax7- Interstitial Cells (PICs) Improves Cardiac Function and Attenuates Remodeling in Mice Subjected to Myocardial Infarction. Cells2022, 11, 4050.

In the original publication [...].

A protocol for extracting immunolabeled murine cardiomyocytes of high-quality RNA by laser capture microdissection.

We developed a highly efficient, ultrashort immunohistochemistry-laser capture microdissection (IHC-LMD) protocol, which allows microdissection of up to 250 single cardiomyocytes. Before LMD, murine hearts are excised, snap-frozen, and cryosectioned. RNA isolated from LMD material is of high RNA quality, making it usable for gene expression analysis and RNA sequencing. Challenges and limitations of this protocol include visualization of the immunostaining and nuclei DAPI dye on the PEN slides, and timing and speed to limit RNA degradation as much as possible.

Transplantation of Skeletal Muscle-Derived Sca-1+/PW1+/Pax7- Interstitial Cells (PICs) Improves Cardiac Function and Attenuates Remodeling in Mice Subjected to Myocardial Infarction.

We have previously shown that skeletal muscle-derived Sca-1+/PW1+/Pax7- interstitial cells (PICs) are multi-potent and enhance endogenous repair and regeneration. Here, we investigated the regenerative potential of PICs following intramyocardial transplantation in mice subjected to an acute myocardial infarction (MI). MI was induced through the ligation of the left anterior descending coronary artery in 8-week old male C57BL/6 mice. 5 × 105 eGFP-labelled PICs (MI + PICs; n = 7) or PBS (MI-PBS; n = 7) were injected intramyocardially into the border zone. Sham mice (n = 8) were not subjected to MI, or the transplantation of PICs or PBS. BrdU was administered via osmotic mini-pump for 14 days. Echocardiography was performed prior to surgery (baseline), and 1-, 3- and 6-weeks post-MI and PICs transplantation. Mice were sacrificed at 6 weeks post-MI + PICs transplantation, and heart sections were analysed for fibrosis, hypertrophy, engraftment, proliferation, and differentiation of PICs. A significant (p < 0.05) improvement in ejection fraction (EF) and fractional shortening was observed in the MI-PICs group, compared to MI + PBS group at 6-weeks post MI + PICs transplantation. Infarct size/fibrosis of the left ventricle significantly (p < 0.05) decreased in the MI-PICs group (14.0 ± 2.5%), compared to the MI-PBS group (32.8 ± 2.2%). Cardiomyocyte hypertrophy in the border zone significantly (p < 0.05) decreased in the MI-PICs group compared to the MI-PBS group (330.0 ± 28.5 µM2 vs. 543.5 ± 26.6 µm2), as did cardiomyocyte apoptosis (0.6 ± 0.9% MI-PICs vs. 2.8 ± 0.8% MI-PBS). The number of BrdU+ cardiomyocytes was significantly (p < 0.05) increased in the infarct/border zone of the MI-PICs group (7.0 ± 3.3%), compared to the MI-PBS group (1.7 ± 0.5%). The proliferation index (total BrdU+ cells) was significantly increased in the MI-PICs group compared to the MI-PBS group (27.0 ± 3.4% vs. 7.6 ± 1.0%). PICs expressed and secreted pro-survival and reparative growth factors, supporting a paracrine effect of PICs during recovery/remodeling. Skeletal muscle-derived PICs show significant reparative potential, attenuating cardiac remodelling following transplantation into the infarcted myocardium. PICs can be easily sourced from skeletal muscle and therefore show promise as a potential cell candidate for supporting the reparative and regenerative effects of cell therapies.

Alterations in the Oral Microbiome Associated With Diabetes, Overweight, and Dietary Components.

The Mediterranean diet (MedDiet) represents the traditional food consumption patterns of people living in countries bordering the Mediterranean Sea and is associated with a reduced incidence of obesity and type-2 diabetes mellitus (T2DM). The objective of this study was to examine differences in the composition of the oral microbiome in older adults with T2DM and/or high body mass index (BMI) and whether the microbiome was influenced by elements of a MedDiet. Using a nested case-control design individuals affected by T2DM were selected from the Seniors-ENRICA-2 cohort concurrently with non-diabetic controls. BMI was measured, a validated dietary history taken, and adherence to a Mediterranean diet calculated using the MEDAS (Mediterranean Diet Adherence Screener) index. Oral health status was assessed by questionnaire and unstimulated whole mouth saliva was collected, and salivary flow rate calculated. Richness and diversity of the salivary microbiome were reduced in participants with T2DM compared to those without diabetes. The bacterial community structure in saliva showed distinct "signatures" or "salivatypes," characterized by predominance of particular bacterial genera. Salivatype 1 was more represented in subjects with T2DM, whilst those with obesity (BMI ≥ 30 kg/m2) had a predominance of salivatype 2, and control participants without T2DM or obesity had an increased presence of salivatype 3. There was an association of salivatype 1 with increased consumption of sugary snacks combined with reduced consumption of fish/shellfish and nuts. It can be concluded that the microbial community structure of saliva is altered in T2DM and obesity and is associated with altered consumption of particular food items. In order to further substantiate these observations a prospective study should be undertaken to assess the impact of diets aimed at modifying diabetic status and reducing weight.

Inhibition of Aurora Kinase B activity disrupts development and differentiation of salivary glands.

Little is known about the key molecules that regulate cell division during organogenesis. Here we determine the role of the cell cycle promoter aurora kinase B (AURKB) during development, using embryonic salivary glands (E-SGs) as a model. AURKB is a serine/threonine kinase that regulates key events in mitosis, which makes it an attractive target for tailored anticancer therapy. Many reports have elaborated on the role of AURKB in neoplasia and cancer; however, no previous study has shown its role during organ development. Our previous experiments have highlighted the essential requirement for AURKB during adult exocrine regeneration. To investigate if AURKB is similarly required for progression during embryonic development, we pharmacologically inhibited AURKB in developing submandibular glands (SMGs) at embryonic day (E)13.5 and E16.5, using the highly potent and selective drug Barasertib. Inhibition of AURKB interfered with the expansion of the embryonic buds. Interestingly, this effect on SMG development was also seen when the mature explants (E16.5) were incubated for 24 h with another cell cycle inhibitor Aphidicolin. Barasertib prompted apoptosis, DNA damage and senescence, the markers of which (cleaved caspase 3, γH2AX, SA-ßgal and p21, respectively), were predominantly seen in the developing buds. In addition to a reduction in cell cycling and proliferation of the epithelial cells in response to AURKB inhibition, Barasertib treatment led to an excessive generation of reactive oxygen species (ROS) that resulted in downregulation of the acinar differentiation marker Mist1. Importantly, inhibition of ROS was able to rescue this loss of identity, with Mist1 expression maintained despite loss of AURKB. Together, these data identify AURKB as a key molecule in supporting embryonic development and differentiation, while inhibiting senescence-inducing signals during organogenesis.

Salivary glands require Aurora Kinase B for regeneration after transient innate immune-mediated injury.

Severe, irreversible salivary gland disease and oral dryness is experienced by sufferers of Sjögren's syndrome and those treated with irradiation for head and neck cancer. Therefore, major efforts have been made in the last decade to unravel key molecular signals that can drive salivary gland (SG) regeneration and functional restoration. However, the earliest molecular determinants that accompany SG regeneration remain incompletely defined. The present study examined the initial mitogenic events marking the regenerative response of the murine submandibular gland (SMG), following innate immune-mediated injury. Local intraductal administration of the synthetic double stranded (ds) RNA polyinosinic-polycytidylic acid (poly (I:C)) widely, but transiently, depleted the acinar and progenitor cells, 24 hours post poly (I:C) introduction. While the progenitor and duct cells started to proliferate and expand at 72 hours, the Mist1-positve acinar cells did not re-appear until 96 hours post poly (I:C) injury. The cellular replenishment during regeneration involved significant upregulation of the cell cycle promoter Aurora kinase B (AURKB). AURKB, which is expressed in healthy proliferating and cancerous cells, is a serine/threonine protein kinase, well known to orchestrate key events in cell division and cytokinesis. However, the expression and role of AURKB in regeneration of post mitotic salivary gland cells has not been previously explored. In vivo inhibition of AURKB using the selective inhibitor Barasertib (AZD1152-HQPA) interfered with SMG recovery from the transient, but severe poly (I:C)-mediated injury and cellular depletion. AURKB deficiency during regeneration of the injured tissues: disrupted cell cycle progression, repressed renewal of Mist1-positive acinar cells and prevented recovery of salivary secretion. The knowledge gained in this study may be utilized in the development of therapeutic targets for irreversible salivary gland disease.

Caspases are key regulators of inflammatory and innate immune responses mediated by TLR3 in vivo.

Understanding the key regulators which impact the innate immune response during initial phases of tissue injury, can advance the use of therapeutic approaches which aim at attenuating inflammation and organ damage. Recognition of microbial components by TLRs, initiates the transcription of innate immune signal pathways, that induce the expression of key inflammatory mediators: cytokines, chemokines and adhesion molecules. Beside regulating apoptotic cell death, recent studies have revealed distinct roles for caspases in the optimal production of inflammatory cytokines and host defense against injurious infections. Whether caspases can play an immune regulatory role in vivo has not been sufficiently investigated. This study aims to explore whether the pan caspase inhibitor z-VAD-fmk can control inflammation and cytokine production subsequent to challenging the innate immunity of the exocrine secretory tissues in vivo. Submandibular glands (SMGs) of the C57BL/6 mice were challenged with the TLR3 stimulant: polyinosinic-polycytidylic acid (poly (I:C)). Results obtained from the current study provide evidence that caspases can control immune responses downstream of TLR3 ligation. The present work proposes a novel mechanism that can prevent overactivation of the innate immunity, which typically leads to fatal immune disorders.

Dysregulation of NF-kB in glandular epithelial cells results in Sjögren's-like features.

The autoimmune disease primary Sjögren's syndrome (pSS) is characterized by hypofunction of the salivary glands (SGs), the cause of which is not correlated to lymphocytic SG infiltration, as prevailing dogma often states. We knocked out the NF-κB proinflammatory pathway inhibitor A20 in keratin14+ epithelial cells, to investigate if immune activated epithelial cells are capable of initiating pSS SG hallmarks. We show that immune activated epithelial cells can cause T cell dominated leukocytic infiltration and immune foci development of the SGs, reflecting the early clinical picture. Infiltrating leukocytes invaded striated ducts, similar to early stage lymphoepithelial lesions observed clinically. Expression of proinflammatory cyto-/chemokines IFNÉ£, TNFα, IL-6, CXCL10 and CXCL13 increased in A20-/- SGs, and functionally both volume and mucin 10 content of whole stimulated saliva from A20-/- mice was significantly reduced. Epithelial cells may therefore represent the initial trigger for pSS SG pathologies, as opposed to simple reactionaries to pre-existing stimuli.

Measurement of intracellular calcium of submandibular glands using a high throughput plate reader.

Calcium ions (Ca2+) impact nearly every aspect of cellular life and intracellular calcium [Ca2+]i is a critical factor in the regulation of a plethora of physiological functions, including: muscle contraction, saliva secretion, metabolism, gene expression, cell survival and death. By measuring the changes of [Ca2+]i levels, critical physiologic functions can be characterized and aberrant pathologic conditions or drug responses can be efficiently monitored. We developed a protocol for assessment of Ca2+ signaling in the acinar units of submandibular glands isolated from C57BL/6 mice, using benchtop, multi-mode, high throughput plate reader (FlexStation 3). This method represents a powerful tool for unlimited in vitro studies to monitor changes in receptor-mediated Ca2+ responses while retaining functional and morphological features of a native setting.