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Rationale: MUC5AC (mucin 5AC, oligomeric gel-forming) and MUC5B (mucin 5B, oligomeric gel-forming) are the predominant secreted polymeric mucins in mammalian airways. They contribute differently to the pathogenesis of various muco-obstructive and interstitial lung diseases, and their genes are separately regulated, but whether they are packaged together or in separate secretory granules is not known. Objectives: To determine the packaging of MUC5AC and MUC5B within individual secretory granules in mouse and human airways under varying conditions of inflammation and along the proximal-distal axis. Methods: Lung tissue was obtained from mice stimulated to upregulate mucin production by the cytokines IL-1 β and IL-13 or by porcine pancreatic elastase. Human lung tissue was obtained from donated normal lungs, biopsy samples of transplanted lungs, and explanted lungs from subjects with chronic obstructive pulmonary disease. MUC5AC and MUC5B were labeled with antibodies from different animal species or, in mice only, by transgenic chimeric mucin-fluorescent proteins and imaged using widefield deconvolution or Airyscan fluorescence microscopy. Measurements and Main Results: In both mouse and human airways, most secretory granules contained both mucins interdigitating within the granules. Smaller numbers of granules contained MUC5B alone, and even fewer contained MUC5AC alone. Conclusions: MUC5AC and MUC5B are variably stored both in the same and in separate secretory granules of both mice and humans. The high fraction of granules containing both mucins under a variety of conditions makes it unlikely that their secretion can be differentially controlled as a therapeutic strategy. This work also advances knowledge of the packaging of mucins within secretory granules to understand mechanisms of epithelial stress in the pathogenesis of chronic lung diseases.

Dry eye disease (DED) is a multifactorial disorder in which the eyes respond to minor stimuli with abnormal sensations, such as dryness, blurring, foreign body sensation, discomfort, irritation, and pain. Corneal pain, as one of DED's main symptoms, has gained recognition due to its increasing prevalence, morbidity, and the resulting social burden. The cornea is the most innervated tissue in the body, and the maintenance of corneal integrity relies on a rich density of nociceptors, such as polymodal nociceptor neurons, cold thermoreceptor neurons, and mechano-nociceptor neurons. Their sensory responses to different stimulating forces are linked to the specific expression of transient receptor potential (TRP) channels. TRP channels are a group of unique ion channels that play important roles as cellular sensors for various stimuli. These channels are nonselective cation channels with variable Ca 2+ selectivity. TRP homologs are a superfamily of 28 different members that are subdivided into 7 different subfamilies based on differences in sequence homology. Many of these subtypes are expressed in the eye on both neuronal and non-neuronal cells, where they affect various stress-induced regulatory responses essential for normal vision maintenance. This article reviews the current knowledge about the expression, function, and regulation of TRPs in ocular surface tissues. We also describe their implication in DED and ocular pain. These findings contribute to evidence suggesting that drug-targeting TRP channels may be of therapeutic benefit in the clinical setting of ocular pain., Competing Interests: The company had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Background: Dry eye disease (DED) is a multifactorial disease of the ocular surface accompanied by neurosensory abnormalities. Here, we evaluated the effectiveness of transient receptor potential vanilloid-1 (TRPV1) blockade to alleviate ocular pain, neuroinflammation, and anxiety-like behavior associated with severe DED., Methods: Chronic DED was induced by unilateral excision of the Harderian and extraorbital lacrimal glands of adult male mice. Investigations were conducted at 21 days after surgery. The mRNA levels of TRPV1, transient receptor potential ankyrin-1 (TRPA1), and acid-sensing ion channels 1 and 3 (ASIC1 and ASIC3) in the trigeminal ganglion (TG) were evaluated by RNAscope in situ hybridization. Multi-unit extracellular recording of ciliary nerve fiber activity was used to monitor spontaneous and stimulated (cold, heat, and acid) corneal nerve responsiveness in ex vivo eye preparations. DED mice received topical instillations of the TRPV1 antagonist (capsazepine) twice a day for 2 weeks from d7 to d21 after surgery. The expression of genes involved in neuropathic and inflammatory pain was evaluated in the TG using a global genomic approach. Chemical and mechanical corneal nociception and spontaneous ocular pain were monitored. Finally, anxiety-like behaviors were assessed by elevated plus maze and black and white box tests., Results: First, in situ hybridization showed DED to trigger upregulation of TRPV1, TRPA1, ASIC1, and ASIC3 mRNA in the ophthalmic branch of the TG. DED also induced overexpression of genes involved in neuropathic and inflammatory pain in the TG. Repeated instillations of capsazepine reduced corneal polymodal responsiveness to heat, cold, and acidic stimulation in ex vivo eye preparations. Consistent with these findings, chronic capsazepine instillation inhibited the upregulation of genes involved in neuropathic and inflammatory pain in the TG of DED animals and reduced the sensation of ocular pain, as well as anxiety-like behaviors associated with severe DED., Conclusion: These data provide novel insights on the effectiveness of TRPV1 antagonist instillation in alleviating abnormal corneal neurosensory symptoms induced by severe DED, opening an avenue for the repositioning of this molecule as a potential analgesic treatment for patients suffering from chronic DED.

Endospore formation is used by members of the phylum Firmicutes to withstand extreme environmental conditions. Several recent studies have proposed endospore formation in species outside of Firmicutes, particularly in Rhodobacter johrii and Serratia marcescens , members of the phylum Proteobacteria. Here, we aimed to investigate endospore formation in these two species by using advanced imaging and analytical approaches. Examination of the phase-bright structures observed in R. johrii and S. marcescens using cryo-electron tomography failed to identify endospores or stages of endospore formation. We determined that the phase-bright objects in R. johrii cells were triacylglycerol storage granules and those in S. marcescens were aggregates of cellular debris. In addition, R. johrii and S. marcescens containing phase-bright objects do not possess phenotypic and genetic features of endospores, including enhanced resistance to heat, presence of dipicolinic acid, or the presence of many of the genes associated with endospore formation. Our results support the hypothesis that endospore formation is restricted to the phylum Firmicutes. Importance: Bacterial endospore formation is an important process that allows the formation of dormant life forms called spores. As such, organisms able to sporulate can survive harsh environmental conditions for hundreds of years. Here, we follow up on previous claims that two members of Proteobacteria, Serratia marcescens and Rhodobacter johrii , are able to form spores. We conclude that those claims were incorrect and show that the putative spores in R. johrii and S. marcescens are storage granules and cellular debris, respectively. This study concludes that endospore formation is still unique to the phylum Firmicutes., (Copyright © 2020 Beskrovnaya et al.)

Competing Interests: None

Corneal pain is considered to be a core symptom of ocular surface disruption and inflammation. The management of this debilitating condition is still a therapeutic challenge. Recent evidence supports a role of the opioid system in the management of corneal nociception. However, the functional involvement of the mu opioid receptor (MOR) underlying this analgesic effect is not known. We first investigated the expression of the MOR in corneal nerve fibers and trigeminal ganglion (TG) neurons in control mice and a mouse model of corneal inflammatory pain. We then evaluated the anti-nociceptive and electrophysiological effects of DAMGO ([D-Ala 2 ,N-Me-Phe 4 ,Gly 5 -ol] enkephalin), a MOR-selective ligand. MOR immunoreactivity was detected in corneal nerve fibers and primary afferent neurons of the ophthalmic branch of the TG of naive mice. MOR expression was significantly higher in both structures under conditions of inflammatory corneal pain. Topical ocular administration of DAMGO strongly reduced both the mechanical (von Frey) and chemical (capsaicin) corneal hypersensitivity associated with inflammatory ocular pain. Repeated instillations of DAMGO also markedly reversed the elevated spontaneous activity of the ciliary nerve and responsiveness of corneal polymodal nociceptors that were observed in mice with corneal pain. Finally, these DAMGO-induced behavioral and electrophysiological responses were totally blunted by the topical application of naloxone methiodide, an opioid receptor antagonist. Overall, these results provide evidence that topical pharmacological MOR activation may constitute a therapeutic target for the treatment of corneal pain and improve corneal nerve function to alleviate chronic pain., (Copyright © 2020 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Dry eye disease (DED) is commonly associated with ocular surface inflammation and pain. In this study, we evaluated the effectiveness of repeated instillations of transient receptor potential melastatin 8 (TRPM8) ion channel antagonist M8-B on a mouse model of severe DED induced by the excision of extra-orbital lacrimal and Harderian glands. M8-B was topically administered twice a day from day 7 until day 21 after surgery. Cold and mechanical corneal sensitivities and spontaneous ocular pain were monitored at day 21. Ongoing and cold-evoked ciliary nerve activities were next evaluated by electrophysiological multi-unit extracellular recording. Corneal inflammation and expression of genes related to neuropathic pain and inflammation were assessed in the trigeminal ganglion. We found that DED mice developed a cold allodynia consistent with higher TRPM8 mRNA expression in the trigeminal ganglion (TG). Chronic M8-B instillations markedly reversed both the corneal mechanical allodynia and spontaneous ocular pain commonly associated with persistent DED. M8-B instillations also diminished the sustained spontaneous and cold-evoked ciliary nerve activities observed in DED mice as well as inflammation in the cornea and TG. Overall, our study provides new insight into the effectiveness of TRPM8 blockade for alleviating corneal pain syndrome associated with severe DED, opening a new avenue for ocular pain management.

The organization of the normal airway mucus system differs in small experimental animals from that in humans and large mammals. To address normal murine airway mucociliary clearance, Alcian blue-stained mucus transport was measured ex vivo on tracheal tissues of naïve C57BL/6, Muc5b -/- , Muc5ac -/- , and EGFP-tagged Muc5b reporter mice. Close to the larynx with a few submucosal glands, the mucus appeared as thick bundles. More distally in the trachea and in large bronchi, Alcian blue-stained mucus was organized in cloud-like formations based on the Muc5b mucin. On tilted tissue, the mucus clouds moved upward toward the larynx with an average velocity of 12 µm/s compared with 20 µm/s for beads not associated with clouds. In Muc5ac -/- mice, Muc5b formed mucus strands attached to the tissue surface, while in Muc5b -/- mice, Muc5ac had a more variable appearance. The normal mouse lung mucus thus appears as discontinuous clouds, clearly different from the stagnant mucus layer in diseased lungs.

Background: Dry eye disease (DED) is a multifactorial disease associated with ocular surface inflammation, pain, and nerve abnormalities. We studied the peripheral and central neuroinflammatory responses that occur during persistent DED using molecular, cellular, behavioral, and electrophysiological approaches., Methods: A mouse model of DED was obtained by unilateral excision of the extraorbital lachrymal gland (ELG) and Harderian gland (HG) of adult female C57BL/6 mice. In vivo tests were conducted at 7, 14, and 21 days (d) after surgery. Tear production was measured by a phenol red test and corneal alterations and inflammation were assessed by fluorescein staining and in vivo confocal microscopy. Corneal nerve morphology was evaluated by nerve staining. Mechanical corneal sensitivity was monitored using von Frey filaments. Multi-unit extracellular recording of ciliary nerve fiber activity was used to monitor spontaneous corneal nerve activity. RT-qPCR and immunostaining were used to determine RNA and protein levels at d21., Results: We observed a marked reduction of tear production and the development of corneal inflammation at d7, d14, and d21 post-surgery in DED animals. Chronic DE induced a reduction of intraepithelial corneal nerve terminals. Behavioral and electrophysiological studies showed that the DED animals developed time-dependent mechanical corneal hypersensitivity accompanied by increased spontaneous ciliary nerve fiber electrical activity. Consistent with these findings, DED mice exhibited central presynaptic plasticity, demonstrated by a higher Piccolo immunoreactivity in the ipsilateral trigeminal brainstem sensory complex (TBSC). At d21 post-surgery, mRNA levels of pro-inflammatory (IL-6 and IL-1β), astrocyte (GFAP), and oxidative (iNOS2 and NOX4) markers increased significantly in the ipsilateral trigeminal ganglion (TG). This correlated with an increase in Iba1, GFAP, and ATF3 immunostaining in the ipsilateral TG of DED animals. Furthermore, pro-inflammatory cytokines (IL-6, TNFα, IL-1β, and CCL2), iNOS2, neuronal (ATF3 and FOS), and microglial (CD68 and Itgam) markers were also upregulated in the TBSC of DED animals at d21, along with increased immunoreactivity against GFAP and Iba1., Conclusions: Overall, these data highlight peripheral sensitization and neuroinflammatory responses that participate in the development and maintenance of dry eye-related pain. This model may be useful to identify new analgesic molecules to alleviate ocular pain.

Objectives: Surfactant protein-D (SP-D), an innate immune defence molecule of the collectin family, is expressed in lungs and additional extrapulmonary epithelia. SP-D has immune modulatory and anti-microbial effects depending on its oligomerization. The ratio of high molecular weight (HMW): low molecular weight (LMW) SP-D in serum is mainly determined by the Met11Thr polymorphism (SNP rs721917). We aimed to study the SP-D serum level and the molecular size distribution in patients with untreated axial spondyloarthritis (axSpA) as compared with control subjects., Methods: Thirty-four patients with disease modifier untreated axSpA according to the ASAS criteria, age 19-63 years, disease duration 3.9 (2.2-5.6) years were included. Demographics, smoking habits, HLA-B27 status, ASDAS, BASDAI, BASFI, BASMI and visual analogue scale scores were recorded. SP-D in serum was measured by ELISA. DNA was isolated from whole blood and single nucleotide polymorphism rs721917 was genotyped. SP-D molecular size distribution was determined using gel filtration chromatography., Results: SP-D in serum did not differ between patients with axSpA and healthy controls, 1177 (869, 1536) vs 910 (494, 1682) (P = 0.35) and SP-D did not correlate with disease activity. However, the HMW/LMW ratio of SP-D in serum was significantly lower in axSpA, 0.38 (0.18, 0.53) compared with controls 1.49 (0.37, 3.24) when adjusting for the Met11Thr polymorphism, gender, age, BMI and smoking (P = 0.0004). There was no correlation between HMW/LMW ratio and CRP or composite diseases outcome measures., Conclusion: We suggest that predominance of LMW oligomeric variants of SP-D may enhance local or systemic inflammatory responses in axSpA.

The respiratory tract is normally kept essentially free of bacteria by cilia-mediated mucus transport, but in chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF), bacteria and mucus accumulates instead. To address the mechanisms behind the mucus accumulation, the proteome of bronchoalveolar lavages from COPD patients and mucus collected in an elastase-induced mouse model of COPD was analyzed, revealing similarities with each other and with the protein content in colonic mucus. Moreover, stratified laminated sheets of mucus were observed in airways from patients with CF and COPD and in elastase-exposed mice. On the other hand, the mucus accumulation in the elastase model was reduced in Muc5b-KO mice. While mucus plugs were removed from airways by washing with hypertonic saline in the elastase model, mucus remained adherent to epithelial cells. Bacteria were trapped on this mucus, whereas, in non-elastase-treated mice, bacteria were found on the epithelial cells. We propose that the adherence of mucus to epithelial cells observed in CF, COPD, and the elastase-induced mouse model of COPD separates bacteria from the surface cells and, thus, protects the respiratory epithelium.

Background and Objective: A structural single nucleotide polymorphism rs721917 in the surfactant protein D (SP-D) gene, known as Met11Thr, was reported to influence the circulating levels and degree of multimerization of SP-D and was associated with both COPD and atopy in asthma. Moreover, disease-related processes are known to degrade multimerized SP-D, however, the degree of the protein degradation in these diseases is not clarified. We aimed to determine the distribution of multimerized (high molecular weight (HMW)) and non-multimerized (low molecular weight (LMW)) species of serum SP-D and their correlation with genetic polymorphisms and presence of disease in Lebanese COPD and asthmatic patients., Methods: Serum SP-D levels were measured by ELISA in 88 COPD, 121 asthmatic patients and 223 controls. Randomly selected subjects were chosen for genotyping of rs721917 and multimerization studies. HMW and LMW SP-D were separated by gel permeation chromatography., Results: Serum SP-D levels were significantly increased in patients with COPD, but not in asthmatic patients, when compared to controls. Met11Thr variation strongly affected serum SP-D levels and the degree of multimerization, but was not associated with COPD and asthma in the study. Remarkably, HMW/LMW serum SP-D ratio was significantly lower in Met11/Met11 COPD and asthmatic patients compared to controls., Conclusion: Collectively, non-multimerized species of serum SP-D were dominant in COPD and asthmatic patients suggesting that degradation of SP-D takes place to a significant degree in pulmonary disease. Assays that can separate SP-D proteolytic breakdown products or modified forms from naturally occurring SP-D trimers may result in optimal disease markers for pulmonary inflammatory diseases., (© 2017 Asian Pacific Society of Respirology.)

Background: SP-D is an important host defense lectin in innate immunity and SP-D deficient mice show several abnormal immune effects and are susceptible to allergen-induced airway disease. At the same time, host microbiome interactions play an important role in the development of allergic airway disease, and alterations to gut microbiota have been linked to airway disease through the gut-lung axis. Currently, it is unknown if the genotype ( Sftpd-/- or Sftpd+/+ ) of the standard SP-D mouse model can affect the host microbiota to such an degree that it would overcome the cohousing effect on microbiota and interfere with the interpretation of immunological data from the model. Generally, little is known about the effect of the SP-D protein in itself and in combination with airway disease on the microbiota. In this study, we tested the hypothesis that microbiome composition would change with the lack of SP-D protein and presence of allergic airway disease in the widely used SP-D-deficient mouse model., Results: We describe here for the first time the lung and gut microbiota of the SP-D mouse model with OVA induced allergic airway disease. After the challenge animals were killed and fecal samples were taken from the caecum and lungs were subjected to bronchoalveolar lavage for comparison of gut and lung microbiota by Illumina 16S rRNA gene sequencing. A significant community shift was observed in gut microbiota after challenge with OVA. However, the microbial communities were not significantly different between SP-D deficient and wild type mice from the same cages in either naïve or OVA treated animals. Wild type animals did however show the largest variation between mice., Conclusions: Our results show that the composition of the microbiota is not influenced by the SP-D deficient genotype under naïve or OVA induced airway disease. However, OVA sensitization and pulmonary challenge did alter the gut microbiota, supporting a bidirectional lung-gut crosstalk. Future mechanistic investigations of the influence of induced allergic airway disease on gut microbiota are warranted.

Biological markers can help to better identify a disease or refine its diagnosis. In the present study, the association between surfactant protein D (SP-D) and chronic obstructive pulmonary disease (COPD) was studied among subjects consulting for respiratory diseases or symptoms and was compared with C-reactive protein (CRP) and fibrinogen. A further aim of this study was to identify the optimal cut-off point of SP-D able to discriminate COPD patients. A case-control study including 90 COPD patients, 124 asthma patients and 180 controls was conducted. Standardized questionnaires were administered and lung function tests were performed. Biological markers were measured in blood samples according to standardized procedures. The association between SP-D and COPD was investigated using logistic regression models. Receiver-operating characteristic curves were used for threshold identification. SP-D levels above the median value were positively associated with COPD [adjusted odds ratio (OR)=3.86, 95% confidence interval (CI): 1.51-9.85, P=0.005). No associations with COPD or asthma were found for CRP or fibrinogen levels. Scores for COPD diagnosis in all COPD patients or ever-smoker COPD patients were identified (sensitivity, 76.4 and 77.8%; specificity, 89.3 and 88.5%, respectively). The results indicate that SP-D can differentiate COPD from other respiratory symptoms or diseases. Used with socio-demographic characteristics and respiratory symptoms, SP-D is able to discriminate COPD patients from controls, particularly among smokers.

Surfactant protein D (SP-D) is a pulmonary collectin important in lung immunity. SP-D-deficient mice (Sftpd(-/-)) are reported to be susceptible to ovalbumin (OVA)- and fungal allergen-induced pulmonary inflammation, while treatment with exogenous SP-D has therapeutic effects in such disease models. β-Glucans are a diverse group of polysaccharides previously suggested to serve as fungal ligands for SP-D. We set out to investigate if SP-D could interact with 1,3-β-glucan and attenuate allergic pulmonary inflammation in the presence of 1,3-β-glucan. Allergic airway disease was induced in Sftpd(-/-) and Sftpd(+/+) mice by OVA sensitization and subsequent challenge with OVA, 1,3-β-glucan, or OVA/1,3-β-glucan together. Mice in the combined treatment group were further treated with a high dose of recombinant fragment of human SP-D (rfhSP-D). We demonstrated direct interaction between SP-D and 1,3-β-glucan. OVA-induced mucous cell metaplasia was increased in Sftpd(-/-) mice, supporting previously reported protective effects of endogenous SP-D in allergy. OVA-induced parenchymal CCL11 levels and eosinophilic infiltration in bronchoalveolar lavage were unaffected by 1,3-β-glucan, but were reversed with rfhSP-D treatment. 1,3-β-Glucan treatment did, however, induce pulmonary neutrophilic infiltration and increased TNF-α levels in bronchoalveolar lavage, independently of OVA-induced allergy. This infiltration was also reversed by treatment with rfhSP-D. 1,3-β-Glucan reduced OVA-induced mucous cell metaplasia, T helper 2 cytokines, and IFN-γ production. rfhSP-D treatment further reduced mucous metaplasia and T helper 2 cytokine secretion to background levels. In summary, rfhSP-D treatment resulted in attenuation of both allergic inflammation and 1,3-β-glucan-mediated neutrophilic inflammation. Our data suggest that treatment with high-dose SP-D protects from mold-induced exacerbations of allergic asthma., (Copyright © 2015 the American Physiological Society.)