The weather in Bloomfield, New Jersey during the winter months often aren't conducive to exercising outside. There are alternatives, such as taking your workout indoors. For those who still want to run or jog outside, you have to learn how to exercise as it gets colder. Depending on the temperature, dressing for the workout is top priority. There's a huge difference in how you dress if the weather is barely over freezing and far below the freezing point. There's also a difference in how your body handles the cold weather.
Your body has to deal with more than just the workout in cold weather.
Cold weather causes the arteries to constrict, which reduces the blood flow to the heart. If you have heart problems, bring your workout indoors. For all others, it can be tougher to get a good workout, but also produces better results when it comes to weight loss. If you have problems with asthma or breathing, don't forget to cover your face with a scarf or ski mask. Bronchioconstriction can occur from the colder temperature and dryer air. The scarf or mask will help moisten the air by trapping the moisture when you exhale. Breathing in through the nose is also important.
Layer your clothing to stay the warmest.
Not only do the layers provide more insulation than one much heavier article of clothing, you can also adjust the layers as the exercise makes you warmer. Sweating in cold weather only adds to the potential of chilling. Don't forget the extremities with warm socks and gloves. Many people recommend the first layer should be of material like thinsulate or other ones made specifically for the cold. Don't forget a hat. You lose lots of body heat through the top of your head. In fact, it's more than any other part of the body.
Check more than just the temperature before going out for a run or jog.
The wind-chill is particularly important in the winter. It causes cold weather to feel even colder. If you've ever run or walked in cold windy weather and had to turn your back on the wind, you know the difference in how cold it is running or walking into the wind compared to having the wind at your back.
Don't forget to do a warm up before starting. You can start inside with dynamic stretches. Take a few extra minutes of warm up time to ensure you have full range of motion. Cold weather brings on stiffness. One way to warm up your lower body is climbing several flights of steps.
Stay hydrated. You may not feel like you're thirsty as you might in the summer, but working out can make you lose fluid in the form of sweat and the air is dryer. If cold water sounds a bit too chilly for you, try some warm tea or even warm water with a bit of lime in it.
Watch out for frostbite. Even though it tends to occur mostly when the temperature is below 5 degrees, wind-chill makes all the difference. Check the windchill before venturing out. At a negative 18 degrees, frostbite occurs within thirty minutes. When they say it feels like -10, it means it affects your body like a -10 degree temperature.
Create an indoor workout or go to the gym on those days that are either too cold, windy or wet for a healthy workout outside.


text:
Human Longevity sues J. Craig Venter Institute, alleging theft of trade secrets
By Bradley J. Fikes
July 23, 2018 8:40 AM PT
This story has been corrected to indicate J. Craig Venter has not been personally sued by HLI.
J. Craig Venter, the San Diego genetic researcher whose drive and insight sped the sequencing of the human genome, has been accused of stealing trade secrets by a company he helped found to exploit one of the great advances in modern science
Human Longevity, Inc., or HLI of La Jolla made the allegation in a lawsuit filed against the J. Craig Venter Institute, which is located a short distance away at UC San Diego.
Venter himself was not sued.
The lawsuit says Venter, 71, made the trade secrets available to competitors of HLI, which is using advanced genomics to search for ways to treat a variety of diseases and disorders and help people to live longer lives.
HLI filed the lawsuit on Friday in the U.S. District Court for the Southern District of California against the institute, or JCVI, and unnamed individual defendants, whose identities aren't yet known.
The company alleges that Venter took the trade secrets after he left the company in late May. Venter had been serving as the chief executive officer. He says he retired. HLI says he was fired.
The lawsuit asks for unspecified damages to be determined at trial, and an injunction preventing misuse of HLI's intellectual property.
Through attorney Steven M. Strauss, JCVI said HLI's claims are "baseless, without merit, and contain numerous factual errors." The response didn't specify any of the errors.
"HLI is one of many companies to have been spun out of the not-for-profit research efforts of the J. Craig Venter Institute and its founder, J. Craig Venter, a renowned genomics pioneer who remains a shareholder in HLI. We intend to vigorously defend against these allegations as the legal process advances."
In July, Venter was replaced on an interim basis by Dr. David Karow, along with Scott Sorensen, interim chief operating officer.
Earlier in May, Venter had withheld critical information on the conduct of a "key HLI executive," that would have resulted in the executive's firing, the lawsuit says. Moreover, Venter appointed an interim president without talking to HLI's board of directors. Venter also had asked for a "one-sided" employment contract, giving him financial advantages.
"Following Venter's termination from HLI, he abruptly left the HLI corporate offices with his HLI-owned computer," the suit states. "Venter immediately began using the HLI computer to communicate to the public, solicit HLI investors and employees."
The purpose of this was to set up a competitor to HLI, using its trade secrets, soliciting investors and hiring away HLI employees, the lawsuit says. All these activities were prohibited by an agreement Venter signed.
HLI knew this because the computer, a Lenovo laptop, was still connected to HLI's server. Messages sent through the computer included Venter's resignation announcement, made on Twitter that day.
On May 25, HLI disabled the computer's access to its email system, but the company still had access to it through a crash recovery program and its email software. HLI says Venter continued to use the Lenovo ThinkPad laptop to distribute proprietary company information.
Human Longevity uses a variety of modern technologies such as genome sequencing and medical imaging to better understand an individual's current health status, risk of disease, and the best methods to maintain health.
Besides this information, HLI retains confidential information on financing sources, along with records of customers and potential customers. These include "high net-worth individuals such as Hollywood actors and actresses, corporate executives, NFL team owners, philanthropists and politicians," the lawsuit said.
Venter is a UC San Diego graduate who co-founded HLI in 2013. The for-profit company is an extension of his life-long efforts to map and explore the human genome. It adds other modern technologies to assess health.
JCVI was founded in 2006 from several predecessor organizations as a non-profit to explore the frontiers of life. Venter has split his time between JCVI, HLI and other companies.
Venter is particularly well known for his efforts in the 1990s to speed up the process of sequencing the human genome. His research placed pressure on the federal government to sharpen and accelerate its own efforts to do the same thing.
In June 2000, President Clinton announced that scientists had completed a draft of the genome, based on work by researchers like Venter, and those by Francis Collins, who led the publicly-funded genome effort.
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bradley.fikes@sduniontribune.com
8:40 a.m. July 23: This article was updated with additional details.
This article was originally published July 22 at 6:00 a.m.
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redpajama_set_name: RedPajamaCommonCrawl


text:
Richmond Guardian (Vic. : 1884 - 1885; 1894 - 1895; 1902 - 1924)
Some Splendid Exhibitions were Given by the Competing Champions.
Australian Ladies' Swimming Championships held in Melbourne this Year The contests to decide the Australian Championship for the year among lady swimmers were commenced at Brunswick on Saturday under the auspices of the Victorian Ladies' Amateur Swimming Association. Strong teams were sent from the adjoining States. The pictures at the top of the page show the New South Wales team (including Misses Fanny Durack and Mina Wylie), the Queensland team and the Victorian team. ... [ILLUSTRATED]
The Consumers' League of Victoria—Cheaper Food for the People, Their Aim.
The executive of the Consumers' League met at the Temperance Hall on Monday evening. Satisfaction was expressed at the outcome of Mr. ...
(1918, February 16). Richmond Guardian (Vic. : 1884 - 1885; 1894 - 1895; 1902 - 1924), p. 1. Retrieved January 22, 2022, from http://nla.gov.au/nla.news-page8503722
Richmond Guardian (Vic. : 1884 - 1885; 1894 - 1895; 1902 - 1924) 16 February 1918: 1. Web. 22 Jan 2022 <http://nla.gov.au/nla.news-page8503722>.
1918, Richmond Guardian (Vic. : 1884 - 1885; 1894 - 1895; 1902 - 1924), 16 February, p. 1. , viewed 22 Jan 2022, http://nla.gov.au/nla.news-page8503722
{{cite news |url=http://nla.gov.au/nla.news-page8503722 |newspaper=[[Richmond Guardian]] |issue=2162 |location=Victoria, Australia |date=16 February 1918 |accessdate=22 January 2022 |page=1 |via=National Library of Australia}}
Funding for digitisation contributed by State Library of Victoria
Richmond Guardian (Vic. : 1884 - 1885; 1894 - 1895; 1902 - 1924), Sat 16 Feb 1918, Page 1


Zimbabwe will host Bangladesh for a full fledged cricket series on April-May 2013. Bangladesh will play 2 Test, 3 ODIs and finally 2 T20s to finish off the bilateral series. BD vs ZIM series 2013 will hit the track on April 17 with the 1st Test at Harare Sports Club ground. 2nd Test match will start from 25th April on the same ground while the ODI & T20 series will be played at Queens Sports Club, Bulawayo. Three ODI between BD vs ZIM will be played on 3, 5 & 8th May. Bangladesh will finish up the series with two T20s on May 11 & 12.
Mushfiqur Rahim (c & wk), Tamim Iqbal, Jahurul Islam, Mohammad Ashraful, Shakib Al Hasan, Mahmudullah, Mominul Haque, Nasir Hossain, Sohag Gazi, Rubel Hossain, Shahadat Hossain, Shahriar Nafees, Enamul Haque, Robiul Islam, Sajidul Islam.


text:
Preparation of 4-hydroxytamoxifen and induction of PKD1 gene deletion
S. rectivirgula preparation and reagents
S. rectivirgula challenge, bronchoalveolar lavage, lung cell isolation, and determination of alveolitis
PCR genotyping, RT-PCR, and RT–real-time PCR
Western blot assay, ELISA, and multiplex sandwich immunoassay
Lung histology
Flow cytometric analysis
Tamoxifen-inducible systemic deletion of PKD1
PKD1 is essential for activation of MAPKs and NF-κB and expression of cytokines and chemokines in the lung after S. rectivirgula inhalation
Significant contribution of PKD1 to the development of a neutrophilic alveolitis following single time exposure to S. rectivirgula
Indispensable contribution of PKD1 to leukocyte infiltration into the bronchial space following repeated exposures to S. rectivirgula
Effects of PKD1 insufficiency on the cellular composition of ILCs and granuloma formation following repeated exposures to S. rectivirgula
Contribution of PKD1 to Th1- and Th17-related cytokine and chemokine expression
Contribution of Protein Kinase D1 on Acute Pulmonary Inflammation and Hypersensitivity Pneumonitis Induced by Saccharopolyspora rectivirgula
Tae Won Yoon;
Tae Won Yoon
*Integrated Biomedical Science Graduate Program, The University of Tennessee Health Science Center, Memphis, TN;
†Department of Microbiology, Immunology and Biochemistry, The University of Tennessee Health Science Center, Memphis, TN;
Elizabeth A. Fitzpatrick;
Elizabeth A. Fitzpatrick
John D. Snyder;
John D. Snyder
Sangmin Lee;
Sangmin Lee
Young-In Kim;
Young-In Kim
‡Department of Pediatrics, The University of Tennessee Health Science Center, Memphis, TN; and
Chidi Zacheaus;
Chidi Zacheaus
§Department of Pharmaceutical Sciences, The University of Tennessee Health Science Center, Memphis, TN
Ae-Kyung Yi
Address correspondence and reprint requests to: Dr. Ae-Kyung Yi, Department of Microbiology, Immunology and Biochemistry, The University of Tennessee Health Science Center, 858 Madison Avenue, Suite 501C, Memphis, TN 38163. E-mail address: ayi@uthsc.edu
Accepted: February 22 2022
Tae Won Yoon, Elizabeth A. Fitzpatrick, John D. Snyder, Sangmin Lee, Young-In Kim, Chidi Zacheaus, Ae-Kyung Yi; Contribution of Protein Kinase D1 on Acute Pulmonary Inflammation and Hypersensitivity Pneumonitis Induced by Saccharopolyspora rectivirgula. Immunohorizons 1 March 2022; 6 (3): 224–242. https://doi.org/10.4049/immunohorizons.2200017
Protein kinase D1 (PKD1), a ubiquitously expressed serine/threonine kinase, regulates diverse cellular processes such as oxidative stress, gene expression, cell survival, vesicle trafficking, Ag receptor signaling, and pattern recognition receptor signaling. We found previously that exposure to hypersensitivity pneumonitis (HP) inciting Ag Saccharopolyspora rectivirgula leads to the activation of PKD1 in a MyD88-dependent manner in various types of murine cells in vitro and in the mouse lung in vivo. However, it is currently unknown whether PKD1 plays a role in the S. rectivirgula–induced HP. In this study, we investigated contributions of PKD1 on the S. rectivirgula–induced HP using conditional PKD1-insufficient mice. Compared to control PKD1-sufficient mice, PKD1-insufficient mice showed substantially suppressed activation of MAPKs and NF-κB, expression of cytokines and chemokines, and neutrophilic alveolitis after single intranasal exposure to S. rectivirgula. The significantly reduced levels of alveolitis, MHC class II surface expression on neutrophils and macrophages, and IL-17A and CXCL9 expression in lung tissue were observed in the PKD1-insufficient mice repeatedly exposed to S. rectivirgula for 5 wk. PKD1-insuficient mice exposed to S. rectivirgula for 5 wk also showed reduced granuloma formation. Our results demonstrate that PKD1 plays an essential role in the initial proinflammatory responses and neutrophil influx in the lung after exposure to S. rectivirgula and substantially contribute to the development of HP caused by repeated exposure to S. rectivirgula. Our findings suggest that PKD1 can be an attractive new molecular target for therapy of S. rectivirgula–induced HP.
Saccharopolyspora rectivirgula is a thermophilic actinomycetes commonly found in moldy hay and is one of the inciting Ags that causes farmer's lung disease, one of the most common types of hypersensitivity pneumonitis (HP) (1). HP, also known as extrinsic allergic alveolitis, is characterized by inflammation of the interstitium, bronchioles, and alveoli of the lung that can ultimately lead to irreversible fibrosis. It is caused by repetitive inhalation of HP Ags in a variety of environmental settings in susceptible individuals (2). The pathogenic mechanisms underlying initiation of HP and the interplay between the host immune system, the environment, and the causative microorganisms are complex and not fully understood. Following inhalation of HP-causing Ags (e.g., S. rectivirgula), pattern recognition receptors on/in lung-resident innate immune cells are triggered, resulting in the production of TNF-α, IL-6, CXCL2, IL-17, and IFN-γ that contribute to the recruitment of neutrophils and other inflammatory cells into the lung (2–9). Chronic exposure to HP Ags results in fibrosis, which is associated with high morbidity and mortality (1, 10, 11). Thus, identification of host innate immune receptors that are involved in initial detection of HP Ags and careful investigation of signal transduction mediated by those innate immune receptors will provide new and important insight into the pathogenesis of this disease.
The protein kinase D (PKD) family is composed of three structurally related serine/threonine kinases; PKD1 (protein kinase C [PKC]μ), PKD2, and PKD3 (PKCν) (12). PKD family proteins are expressed ubiquitously and implicated in the regulation of a variety of cellular and subcellular processes such as vesicle transportation, reactive oxygen species generation, activation of MAPKs and NF-κB, and regulation of cell shape, motility, adhesion, and gene expression (13–20). PKDs can be activated by upstream signaling modulators such as PI3K, phospholipase Cγ, diacylglycerol, and classical/novel PKCs that are activated through signals transduced by G protein–coupled receptor agonists, reactive oxygen species, epidermal growth factor receptor, FcγRII, BCR, and TCR (13, 20–25). Depending on the cell context and the stimulation conditions, PKDs display dynamic changes in intracellular locations (such as cytosol, the plasma membrane, the mitochondria, the Golgi apparatus, or the nucleus), and cellular location of PKDs has been shown to dictate the biological role of the protein (14, 19, 20, 26–29). PKD family members have also been shown to be involved in innate immunity. All three PKD family members activated by diacylglycerol at the Golgi membrane can interact with NLRP3 and activate NLRP3 inflammasome (30). TLR4 ligand–mediated p38 activation and TNF-α secretion in microglial cells, TLR5 ligand–mediated p38 activation and IL-8 production in epithelial cells, TLR1/2 ligand–induced expression of heat shock protein 27 and MCP-1 in mouse bone marrow–derived mast cells are suppressed by a PKC/PKD inhibitor, Gö6976 (31–33). In addition to these studies, we previously found that PKD1, but not PKD2 and PKD3, is recruited to the TLR/MyD88/IL-1R–associated kinase (IRAK)/TNFR-associated factor 6 (TRAF6) signaling complex and being activated upon the TLR ligand stimulation and required for the MyD88-dependent activation of NF-κB and MAPKs, and subsequent expression of cytokines TNF-α, IL-6, IL-12, chemokine MCP-1, and costimulatory molecule CD86 (34, 35).
TLR2 and TLR9 have been demonstrated to be involved in mediating proinflammatory responses, neutrophil recruitment to the airway, and Th17 development after acute and chronic exposures to an HP inciting the Ag S. rectivirgula (36, 37). The initial proinflammatory responses to S. rectivirgula in the lung, including production of proinflammatory cytokines and chemokines and subsequent neutrophil infiltration, are largely dependent on MyD88, the major signaling adaptor molecule in the TLR/IL-1R signaling pathway (38). These findings support the notion that interruption of the MyD88-dependent signaling pathway at the early stage might have significant beneficial effects on prevention and/or attenuation of the disease progress.
In the MyD88-dependent TLR/IL-1R signaling pathway, the receptor bound MyD88 leads to the recruitment of IRAK family members IRAK4 and IRAK1, PKD1, and an E3 ubiquitinase TRAF6 to the TLR/MyD88 signaling complex (32, 34, 35, 39–43). The recruitment to the receptor complex and activation of PKD1 is dependent on IRAK4 and IRAK1 in this pathway (35, 44). In turn, PKD1 is required for the ubiquitination of TRAF6 and subsequent activation of TGF-β–activated kinase 1 (TAK1), which initiates signaling cascades that lead to activation of NF-κB and MAPKs, resulting in expression of proinflammatory cytokines and chemokines (35, 44–48). Previously, we found that S. rectivirgula induces activation of PKD1 in an MyD88-dependent manner in the mouse lung in vivo and in various murine cell lines, including MLE12 (alveolar type II epithelial cells), MPRO (promyelocytes differentiated to neutrophils), RAW264.7 (macrophages), and AMJ2-C11 (alveolar macrophages [AMs]) (49). We also found that S. rectivirgula–mediated production of proinflammatory cytokines and chemokines in these cell lines are significantly suppressed when PKD1 expression is knocked down by PKD1-specific small interfering RNA (49). Furthermore, we demonstrated that S. rectivirgula–induced acute lung injury accompanied by neutrophilic alveolitis and increased proinflammatory cytokines and chemokines are significantly inhibited in mice pretreated with Gö6976, a pharmacological inhibitor that inhibits the activity of PKCα, PKCβ, checkpoint kinases 1 and 2 (CHK1/2), PKD1, PKD2, and PKD3 (49–51). These findings suggest that PKD1 is one of the critical factors required for development of proinflammatory reactions in the lung after inhalation of S. rectivirgula. However, it is currently unknown whether PKD1 plays any biologic role in acute pulmonary proinflammatory responses to inhaled S. rectivirgula and in development of HP. In the current study, we investigated whether PKD1 is essential for mediating acute lung injury after exposure to S. rectivirgula and whether PKD1 contributes to the neutrophil and T cell influx, Th1/Th7 related cytokine and chemokine expression, and granuloma formation during the development of HP using inducible PKD1-insufficient mice.
C57BL/6 mice and Gt(ROSA)26Sortm1(cre/ERT2)Tyj/J (R26Cre/ERT2) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). PKD1fl/fl mice (mice with inserted loxP sites in the PKD1 gene to flanking exons 12–14 that encode the kinase function) were provided by Dr. E. Olson (University of Texas Southwestern Medical Center, Dallas, TX) (52). PKD1fl/fl mice and R26Cre/ERT2 mice were backcrossed onto the C57BL/6 for more than nine generations at The University of Tennessee Health Science Center (UTHSC). Subsequent cross-breeding of these PKD1fl/fl (C57BL/6) and R26Cre/ERT2 (C57BL/6) mice resulted in PKD1fl/fl-R26Cre/ERT2 (C57BL/6), which were bred and maintained in a pathogen-free facility at UTHSC. All animal care and housing requirements set forth by the National Institutes of Health Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources were followed. Animal protocols were reviewed and approved by the UTHSC Institutional Animal Care and Use Committee.
4-Hydroxytamoxifen (4-HT; H6278; Sigma-Aldrich, St. Louis, MO) was dissolved in ethanol (20 mg/ml) and mixed with an equal volume of sunflower oil (Sigma-Aldrich) by vertexing. The residual ethanol was removed by evaporation in a SpeedVac vacuum concentrator. To induce deletion of the PKD1 gene, PKD1fl/fl-R26Cre/ERT2 mice were treated with 100 μl of sunflower oil (wild-type [WT] control, PKD1-sufficient mice) or 100 μl of 4-HT dissolved in sunflower oil (PDK1 knockout [PKD1KO], PKD1-insufficient mice) by i.p. injection on days 0, 1, 3, 4, 6, and 7. To provide a control for 4-HT treatment, PKD1fl/fl mice were also treated with 100 μl of sunflower oil or 100 μl of 4-HT dissolved in sunflower oil by i.p. injection on days 0, 1, 3, 4, 6, and 7. Mice were challenged with S. rectivirgula within 2 wk after the last 4-HT treatment.
S. rectivirgula strain (ATCC 15347) was grown in trypticase soy broth (Sigma-Aldrich) at 55°C in a shaking incubator for 2 d, centrifuged, and washed three times with endotoxin-free water (Sigma-Aldrich). Bacterial cells were lyophilized. The lyophilized bacterial pellet was reconstituted in endotoxin-free saline at a concentration of 10 mg/ml and kept at −80°C until used. Reconstituted S. rectivirgula had no detectable endotoxin by Limulus assay (Sigma-Aldrich).
Mice were exposed intranasally to S. rectivirgula (100 μg/50 μl of endotoxin-free saline) or endotoxin-free saline (50 μl) (AdipoGen Life Sciences, San Diego, CA) for one or three times per week for 5 wk. Mice were euthanized at designated time points (1, 6, 24, 48, or 72 h after the last S. rectivirgula challenge). Unless otherwise indicated, control and S. rectivirgula–exposed mice were analyzed individually. To perform bronchoalveolar lavage (BAL), mice tracheas were cannulated, and the lungs were washed with 1 ml of 2 mM EDTA/PBS. The typical amount of fluid recovered was ∼70% of the input. The recovered BAL fluid (BALF) was centrifuged, and the resulting supernatants were kept at −80°C until use for detection of cytokines and chemokines. The cells recovered from the BALF were counted using trypan blue dye exclusion and used for flow cytometric analysis. Following BAL, the lungs were perfused through the right ventricle with PBS and digested with collagenase (20 U/ml) and DNase I (40 μg/ml) for 45 min at 37°C. Cells were freed by mechanical disruption and filtered through 40-μm nylon mesh. Discontinuous Ficoll-Paque Plus (GE Healthcare Bio-Sciences) gradient centrifugation was used to separate extraneous fibroblasts or epithelial cells. The mononuclear cells were isolated at the 40/80% interface and used for flow cytometric analysis and total cell counts. The degree of alveolitis was determined by counting the number of interstitial lung tissue cells or live cells recovered in BALF using trypan blue exclusion. The cellular composition of the alveolitis was determined by flow cytometric analysis.
To distinguish PKD1 alleles, genomic DNA was isolated using a DNeasy tissue kit (Qiagen, Valencia, CA) and PKD1 alleles were analyzed (52). The sequences of PCR primers used are as follows: WT/loxP (366 forward, 5′-GCCCACAGCTATTGTTCCTAA-3′, 517 reverse, 5′-GGATAAAGTGATCAAGCAGCA-3′) and KO (LA1 forward, 5′-GAGCTTCACTTGGAATGACAC-3′, SA4 reverse, 5′-CCTTTAATCTGAGCACTTGG-3′). For RT-PCR, DNA-free total RNA was isolated and the relative amounts of selected gene transcripts were analyzed by RT-PCR as described previously (44). Actin or GAPDH was used as a loading control for all RT-PCR reactions. Sequences of RT-PCR primers for PKD1 are as follows: forward, 5′-AAGGCACT CTGGAGATTGG-3′, reverse, 5′-ACTTCAGGTGCCAGGTATGC-3′ (as published previously in Ref. 34). Sequences of RT-PCR primers for mouse genes were described previously (34, 35, 44, 49, 53). All primers were purchased from Integrated DNA Technologies (Coralville, IA). For RT–real-time PCR (TaqMan assay and SYBR Green assay), total RNA (250 ng) was reverse transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems). Next, quantitative RT-PCR was performed on an QuantStudio 5 (Applied Biosystems) using the TaqMan universal PCR master mix (Applied Biosystems) or SYBR Green master mix (Applied Biosystems). Primers were designed and supplied by Applied Biosystems (for TaqMan assay) and Integrated DNA Technologies (for SYBR Green assay). The product size was initially monitored by agarose gel electrophoresis. Melting curves were analyzed to control for specificity of PCR reactions. The data on genes that were differentially expressed were normalized to the expression the housekeeping gene, actin. The relative units were calculated from a standard curve, plotting three different concentrations against the PCR cycle number at which the measured intensity reaches a fixed value (with a 10-fold increment equivalent to ∼3.1 cycles). Fold change comparing S. rectivirgula–treated exposed PKD1-sufficient mice and S. rectivirgula–treated PKD1-insufficient mice to control PKD1-sufficient mice exposed to saline were calculated by the comparative quantification algorithm-ΔΔCt method (fold difference = 2−ΔΔCt). Primer information is listed in Table I.
Table I.
List of real-time PCR primers used
Gene .
Chemistry .
Assay ID .
Reference Sequence No. or Primer Sequences .
IL-17A TaqMan Mm00439618_m1 NM_010552.3
IL-17RA TaqMan Mm00434214_m1 NM_008359.2
CXCL9 TaqMan Mm00434946_m1 NM_008599.4
CXCL11 TaqMan Mm00444662_m1 NM_019494.1
CXCL12 TaqMan Mm00445553_m1 NM_001012477.2, NM_013655.4, NM_021704.3
CXCL13 TaqMan Mm01208154_g1 NM_018866.2
CCL20 TaqMan Mm01268754-m1 NM_001159738.1, NM_016960.2
β-Actin TaqMan Mm02619580_g1 GenBank: AK075973.1
IFN-γ SYBR Green Mm.PT.58.41769240 5′-CTGAGACAATGAACGCTACACA-3′
5′-TCCACATCTATGCCACTTGAG-3′
β-Actin SYBR Green Mm.PT.39a.22214843.g 5′-GATTACTGCTCTGGCTCCTAG-3′
5′-GACTCATCGTACTCCTGCTTG-3′
Levels or phosphorylation status of specific proteins in whole-lung tissue extracts were analyzed by Western blot assay as described previously (34, 53). Blots developed in ECL reagents (GE Healthcare, U.K.) were either exposed onto x-ray film followed by processing through an X-OMAT 2000A processor (Kodak) or scanned using a ChemiDoc touch imaging system (Bio-Rad, Hercules, CA). Blots developed with fluorochrome-conjugated secondary Abs were scanned using an Odyssey CLx (LI-COR Biosciences, Lincoln, NE). Actin was used as a loading control for all Western blot assays. All phospho-specific Abs were purchased from Cell Signaling Technology (Beverly, MA). Abs specific for actin, MyD88, TRAF6, and IκBα were purchased from Santa Cruz Biotechnology (Dallas, TX). Abs specific for PKD1 were purchased from Santa Cruz Biotechnology and OriGene (Rockville, MD). Concentrations of IFN-γ, TNF-α, IL-6, IL-12, IL-17A, IL-21, IL-22, and IL-23 in BALF were analyzed by ELISA as described (34, 53). All ELISA kits were purchased from Invitrogen. Concentrations of IL-1α, IL-1β, MCP-1, MIP-2, keratinocyte chemoattractant (KC), IL-2, IL-4, IL-6, and IL-10 in BALF were analyzed using a multiplex sandwich assay kit (MCYTOMAG-70K, EMD Millipore, Billerica, MA) following the manufacturer's protocols. The fluorescence was measured with Bio-Plex MAGPIX multiplex reader (Bio-Rad, Hercules, CA).
The left lung lobes removed from the mice were fixed in 10% neutral buffered formalin, dehydrated, and embedded in paraffin. The paraffin-embedded lungs were sectioned longitudinally at 5 μm and stained with H&E. To determine inflammatory cell influx into the interstitial lung tissue, digital images of whole H&E-stained slides were captured using the Aperio ScanScope XT slide scanner (Aperio Technologies, Vista, CA).
For splenic immune cell profiling, spleen cells were incubated with anti-CD16/CD32 (2.4G2) Abs and subsequently stained with fluorochrome-conjugated Abs to CD45 (I3/2.3), CD19 (6D5), βTCR (H57-597), CD3 (17A2), CD4 (RM4-5), CD8 (53-6.7), and CD11b (M1/70), and then analyzed using a BD Biosciences LSR II flow cytometer (BD Biosciences, San Diego, CA) and FlowJo flow cytometry data analysis software (FlowJo, Ashland, OR). Debris and doublets were gated out (Supplemental Fig. 1A). In the single-cell gate, the CD19+ population was identified as B cells (CD19+) and the CD45+CD11b+ population was identified as CD11b+ cells. In the single-cell gate, the βTCR+CD3+ population was gated, and then the CD4+ population in the βTCR+CD3+ gate was identified as CD4+ T cells (βTCR+CD4+) and the CD8+ population in the βTCR+CD3+ gate was identified as CD8+ T cells (βTCR+CD8+). The frequency of each cell population was expressed as the percentage of spleen cells. To detect neutrophils in the airway after one-time S. rectivirgula exposure, cells recovered from BALF were incubated with anti-CD16/CD32 Abs, subsequently stained with fluorochrome-conjugated Abs to CD11b, F4/80 (BM8), Gr-1(RB6-8C5), and MHC class II (MHC-II) (M5/114.15.2), and then analyzed using a BD Biosciences LSR II flow cytometer and FlowJo flow cytometry data analysis software. Debris and doublets were gated out (Supplemental Fig. 1B). In the single-cell gate, the CD11b+Gr1+ population was gated, and then the Gr1+F4/80− population in the CD11b+Gr1+ gate was identified as neutrophils (CD11b+Gr1+F4/80−). The frequency of CD11b+Gr1+F4/80− cells was expressed as the percentage of BAL cells. To detect neutrophils among interstitial lung cells (ILCs) recovered from the lungs isolated from mice after one-time S. rectivirgula exposure, ILCs were incubated with anti-CD16/CD32 Abs, subsequently stained with fluorochrome-conjugated Abs to CD45, CD11b, F4/80, Gr-1, and MHC-II, and then analyzed using a BD Biosciences LSR II flow cytometer and FlowJo flow cytometry data analysis software. Debris and doublets were gated out (Supplemental Fig. 1C). In the single-cell gate, the CD45+ population was gaited and identified as CD45+ cells. In the CD45+ gate, the CD11b+Gr1+ population was gated, and then the Gr1+F4/80− population in the CD11b+Gr1+ gate was identified as neutrophils (CD45+CD11b+Gr1+F4/80−). Frequency of the CD45+CD11b+Gr1+F4/80− cell population was expressed as the percentage of ILCs. To determine cellular composition of alveolitis following a 5-wk exposure of mice to S. rectivirgula, cells recovered from BALF were incubated with anti-CD16/CD32 Abs, subsequently stained with fluorochrome-conjugated Abs to CD11b, CD11c (N418), F4/80, Gr-1, MHC-II, CD86 (GL-1), βTCR, CD4, CD8, and/or CD69 (H1.2F3), and then analyzed using a BD Biosciences LSR II flow cytometer and FlowJo flow cytometry data analysis software. Debris was excluded (Supplemental Fig. 2A). In the single-cell gate, the CD11b+Gr1+ population was identified as polymorphonuclear neutrophils (PMNs; CD11b+Gr1+), the CD11c+F4/80+ population was identified as AMs (CD11c+F4/80+), the CD4+βTCR+ population was identified as CD4+ T cells (CD4+βTCR+), and the CD8+βTCR+ population was identified as CD8+ T cells (CD8+βTCR+). The frequency of each cell population was expressed as the percentage of BAL cells. The geometric mean fluorescence intensities (gMFIs) of CD86 and MHC-II on PMNs and AMs and of CD69 on CD4+ T cells and CD8+ T cells were calculated using FlowJo flow cytometry data analysis software. To determine cellular composition of ILCs following a 5-wk exposure of mice to S. rectivirgula, ILCs were incubated with anti-CD16/CD32 Abs, subsequently stained with fluorochrome-conjugated Abs to CD11b, CD11c (N418), F4/80, Gr-1, MHC-II, βTCR, CD4, CD8, and/or CD69 (H1.2F3), and then analyzed using a BD Biosciences LSR II flow cytometer and FlowJo flow cytometry data analysis software. Debris was excluded (Supplemental Fig. 2B). In the single-cell gate, the CD11b+CD11c+ population, CD11b+Gr1+ population, CD11b+F4/80+ population, CD11b−CD11c+ population, CD4+βTCR+ population, and CD8+βTCR+ population were identified. The frequency of each cell population was expressed as the percentage of ILCs. The gMFIs of MHC-II on the CD11b+CD11c+ population, CD11b+Gr1+ population, CD11b+F4/80+ population, and CD11b−CD11c+ population, as well as CD69 on the CD4+βTCR+ population and CD8+βTCR+ population, were calculated using FlowJo flow cytometry data analysis software. All Abs were purchased from BioLegend (San Diego, CA) and eBioscience.
Data were expressed as mean values ± SD. The differences between two groups were evaluated using a two-tailed Student t test. The differences between multiple groups were evaluated using a one-way ANOVA with a Tukey post hoc test. GraphPad Prism statistical software (GraphPad Software, San Diego, CA) was used. Statistically significant differences are indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
To induce depletion of the PKD1 gene from the mouse genome, PKD1fl/fl-R26Cre/ERT2 mice were injected i.p. with 4-HT, as indicated in (Fig. 1A. The PKD1 gene deletion was confirmed by the presence of a 359-bp knockout band in the 4-HT–treated PKD1fl/fl-R26Cre/ERT2 mice on PCR analysis of genomic DNA (Fig. 1B, left panel). Expression levels of PKD1 mRNA (Fig. 1B, second panel from left) and protein (Fig. 1B, third panel from left) were substantially inhibited in the lungs of the 4-HT–treated PKD1fl/fl-R26Cre/ERT2 mice. In contrast, levels of mRNA and protein from other genes tested in the lungs of the 4-HT–treated PKD1fl/fl-R26Cre/ERT2 mice were comparable to those in the lungs in the vehicle-treated control mice. To evaluate whether 4-HT treatment or PKD1 deletion affected the general immune cell profile, spleen cells were analyzed by flow cytometry. Proportions of B cells, CD4+ T cells, CD8+ T cells, and CD11b+ cells in the spleens of 4-HT–treated PKD1fl/fl-R26Cre/ERT2 mice were comparable to those of vehicle-treated PKD1fl/fl-R26Cre/ERT2 mice (Fig. 1B, right panel). Of note, 4-HT treatments neither deleted PKD1 nor altered immune cell profile in PKD1fl/fl mice (the strain lacks the Cre recombinase) (Fig. 1C). These data demonstrate that our 4-HT treatment regimen effectively deleted PKD1 in PKD1fl/fl-R26Cre/ERT2 mice and that the 4-HT treatment regimen did not alter the general immune cell profile in mouse spleen.
Induction of PKD1 gene deletion in PKD1fl/fl-R26Cre/ERT2 mice by 4-HT treatment.
(A) 4-HT administration scheme. (B) C57BL/6 mice and PKD1fl/fl-R26Cre/ERT2 mice were treated with vehicle (sunflower oil) or 4-HT six times by i.p. injection. Genomic DNA was isolated from lung and spleen and analyzed for PKD1 alleles by PCR. PCR products corresponding to WT (151 bp), PKD1loxP (255 bp), and PKD1KO (359 bp) are shown (left panel). Total RNA was isolated from lung and mRNA levels of the indicated genes were analyzed by RT-PCR (second panel). Whole-lung lysates were prepared, and levels of the indicated proteins were detected by Western blot assay (third panel). To detect the indicated cell population, cells isolated from spleen were analyzed by flow cytometry as described in Materials and Methods (right panel). Frequency of each cell population is expressed as the percentage of spleen cells. (C) PKD1fl/fl mice were treated with vehicle or 4-HT six times by i.p. injection. Genomic DNA was isolated from lungs and analyzed for PKD1 alleles by PCR. PCR products corresponding to WT (151 bp), PKD1loxP (255 bp), and PKD1KO (359 bp) are shown (left panel). Total RNA was isolated from lung and mRNA levels of the indicated genes were analyzed by RT-PCR (second panel). To detect the indicated cell population, cells isolated from spleen were analyzed by flow cytometry as described in Materials and Methods (right panel). Frequency of each cell population is expressed as the percentage of spleen cells. Each lane represents the samples collected from an individual mouse. For immune cell profiling (right panels in B and C), the number of mice used for each group is as follows: PKD1fl/fl-R26Cre/ERT2 mice-vehicle, n = 2; PKD1fl/fl-R26Cre/ERT2 mice–4-HT, n = 3; PKD1fl/fl mice-vehicle, n = 4; and PKD1fl/fl mice–4-HT, n = 4. All experiments were repeated two to four times with similar results. Veh, vehicle.
We have previously found that S. rectivirgula induces activation of PKD1 in various murine cell lines in vitro and in the lung in vivo and that expression of various proinflammatory cytokines and chemokines in response to S. rectivirgula is significantly suppressed in PKD1-knockdown cells (49). These findings suggested a possibility that PKD1 plays an essential role in the S. rectivirgula–induced pulmonary inflammation. We tested this using tamoxifen-inducible systemic PKD1-insufficient mice. PKD1fl/fl-R26Cre/ERT2 mice were treated with either vehicle or 4-HT as indicated in (Fig. 1A. With these vehicle-treated PKD1fl/fl-R26Cre/ERT2 mice (WT; PKD1-sufficient mice) and 4-HT–treated PKD1fl/fl-R26Cre/ERT2 mice (PKD1KO; PKD1-insufficient mice), we investigated whether PKD1 is essential for activation of MAPKs and NF-κB and expression of key cytokines and chemokines that influence the cytokine milieu and leukocyte influx in the lung following single intranasal exposure to S. rectivirgula. As shown in (Fig. 2A, S. rectivirgula induced activation of PKD1, MAPKs (JNK, ERK, and p38), and degradation of IκBα (as an indication of NF-κB activation) in the lungs of PKD1-sufficient mice (WT) within 1 h of exposure. However, the S. rectivirgula–mediated activation of PKD1, JNK, ERK, and p38 and degradation of IκBα were substantially inhibited in the lungs of PKD1-insufficient mice (PKD1KO). These results indicate that PKD1 plays an indispensable role in S. rectivirgula–induced activation of JNK, ERK, p38, and NF-κB in the lung. In addition, these results further verify the previous finding (49) that S. rectivirgula induces activation of PKD1 among three PKD family members.
PKD1 contributes to the proinflammatory responses in the lung that develop after S. rectivirgula inhalation.
(A–C) PKD1fl/fl-R26Cre/ERT2 mice were treated with vehicle (WT) or 4-HT (PKD1KO) six times by i.p. injection, as described in Materials and Methods. Within 1 wk after the last 4-HT treatment, mice were exposed intranasally to saline or S. rectivirgula for 1 h (A and B), 6 h [for KC and MIP-2 in (C)], or 24 h [for IL-1α, IL-1β, IL-6, IL-12, TNF-α, and MCP-1 in (C)]. (A) Lung lysates were prepared and the activation status of PKD1, JNK, p38, and ERK and protein levels of IκBα and actin in lung lysates were detected by Western blot. Actin was used as a loading control. Each lane represents the samples collected from an individual mouse. (B) Total RNA was purified from lung lobes and mRNA levels of the indicated genes were analyzed by RT-PCR. Each lane represents the samples collected from an individual mouse. GAPDH and actin were used as loading controls. (C) Bronchoalveolar lavage (BAL) was performed. Levels of the indicated cytokines and chemokines in BAL fluid were detected by either ELISA (TNFα, IL-6, and IL-12) or a multiplex sandwich assay (IL-1α, IL-1β, MCP-1, MIP-2, and KC). Data represent the mean concentration (pg/ml) ± SD. The number of mice used for each group is as follows: for 6-h S. rectivirgula challenge with WT-saline, n = 2; WT–S. rectivirgula, n = 4; PKD1KO–S. rectivirgula, n = 4; for 24-h S. rectivirgula challenge with WT-saline, n = 5; WT–S. rectivirgula, n = 6; PKD1KO–S. rectivirgula, n = 9. All experiments were performed two to three times with similar results. *p < 0.05, **p < 0.01, ***p < 0.001. SR, S. rectivirgula.
Next, proinflammatory responses in the lungs of PKD1-sufficient mice and PKD1-insufficient mice exposed to S. rectivirgula were assessed by analyzing mRNA levels and protein levels of selected cytokines and chemokines in lungs and BALF, respectively. As demonstrated in (Fig. 2B, there was increased expression of TNF-α, IL-6, IFN-β, IL-12p40, keratinocyte chemoattractant (KC; CXCL1), LPS-induced CXC chemokine (LIX; CXCL5), MCP-1 (CCL2), MIP-1α (CCL3), MIP-2 (CXCL2), and RANTES (CCL5) in the lungs of PKD1-sufficient mice within an hour after exposure to S. rectivirgula. We were not able to detect expression of eotaxin, CCR5, and CXCR4 in the lungs of these mice within 1 h after S. rectivirgula exposure (data not shown). Although there were slight variations among individuals, when compared with PKD1-sufficient mice, PKD1-insufficient mice showed substantially reduced mRNA expression levels of cytokines TNF-α, IL-6, and IL-12p40 and chemokines KC, LIX, MCP-1, MIP-1α, MIP-2, and RANTES in the lung in response to S. rectivirgula exposure. However, S. rectivirgula–induced expression of IFN-β in the lungs was not inhibited in PKD1-insufficient mice. Protein levels of IL-1α, IL-1β, IL-6, IL-12, TNF-α, KC, MCP-1, and MIP-2 in BALF were significantly increased in PKD1-sufficient mice following exposure to S. rectivirgula (Fig. 2C). Compared to those in PKD1-sufficient mice, protein levels of IL-6, IL-12, TNF-α, MCP-1, and MIP-2 in BALF from S. rectivirgula–exposed PKD1-insufficient mice were significantly decreased, indicating an indispensable role of PKD1 in the S. rectivirgula–induced production of these proinflammatory mediators in the lung. Protein levels of IL-1α, IL-1β, and KC in BALF from S. rectivirgula–exposed PKD1-insufficient mice were not significantly different from those in PKD1-sufficient mice. These results indicate that PKD1 is dispensable for the S. rectivirgula–induced expression of IFN-β and production of IL-1α, IL-1β, and KC in the lung. Of note, mRNA expression levels of cytokines IL-6 and IL-12 in the lungs and BALF protein levels of cytokines TNF-α, IL-6, and IL-12 in response to S. rectivirgula in PKD1fl/fl mice treated with 4-HT were comparable to those in the vehicle-treated PKD1fl/fl mice (Fig. 3A, 3B). These findings demonstrate that the 4-HT treatment regimen used in our experiment does not have immune-suppressive effects. Collectively, these results demonstrated that PKD1 plays an essential role in S. rectivirgula–mediated activation of JNK, ERK, p38, and NF-κB and contributes significantly to the subsequent proinflammatory responses in the lung. Our results also imply that pulmonary proinflammatory responses to S. rectivirgula might involve a complicated orchestration of multiple signaling pathways.
The 4-HT treatment regimen does not have immune suppressive effects.
Control PKD1fl/fl mice (n = 3/group) were treated with vehicle or 4-HT six times by i.p. injection, as described in Materials and Methods. Within 1 wk after the last 4-HT treatment, mice were exposed intranasally to saline or S. rectivirgula for 6 h. (A) Total RNA was purified from lung lobes and mRNA levels of the indicated genes were analyzed by RT-PCR. Each lane represents the samples collected from an individual mouse. GAPDH was used as a loading control. (B) BAL was performed. Levels of IL-6 and IL-12 in BALF were detected by ELISA. Data represent the mean concentration (pg/ml) ± SD. (C) BAL was performed, and the BAL cells were recovered. Cells were counted to determine the degree of alveolitis using trypan blue exclusion. Total neutrophil cell counts were derived by staining BAL cells with Abs to Gr-1, CD11b, and F4/80 followed by flow cytometry as described in Materials and Methods. Neutrophils were identified as CD11b+Gr1+/F4/80−, and the frequency of neutrophils is expressed as the percentage of BAL cells. Data represent the mean cell number ± SD. All experiments were done two to three times with similar results. *p < 0.05; ns, not significant. SR, S. rectivirgula.
Our results showed significant reduction in S. rectivirgula–induced pulmonary expression and production of MIP-2 and MCP-1, chemokines that attract leukocytes into the lungs after S. rectivirgula exposure (38, 54), in PKD1-insufficient mice. These results suggest a possibility that PKD1 may contribute to the neutrophil influx into the lung observed after exposure to S. rectivirgula. Therefore, we further investigated whether deletion of PKD1 also affects leukocyte infiltration into the lung following S. rectivirgula exposure. As expected, at 6 or 24 h after exposure to S. rectivirgula, PKD1-sufficient mice exhibited dramatic increases in total BAL cell numbers (alveolitis) and total ILC numbers compared with control mice exposed to saline (Fig. 4A). Histological sections of lungs from S. rectivirgula–exposed PKD1-sufficient mice also showed the presence of extensive mononuclear cell infiltration in the lungs compared with those from saline-exposed mice (Fig. 4B). In agreement with previous studies (38, 49, 54, 55), neutrophils were the predominant cell type recovered from the airways and lung tissues isolated from PKD1-sufficient mice exposed to S. rectivirgula (Fig. 4A). In contrast, significantly fewer BAL cells and ILCs were recovered from the PKD1-insufficient mice at 6 or 24 h after the S. rectivirgula exposure compared with those from PKD1-sufficient mice (Fig. 4A). Histological sections of lungs from S. rectivirgula–exposed PKD1-insufficient mice also showed significantly less mononuclear cell infiltration in the lungs compared with those from S. rectivirgula–exposed PKD1-sufficient mice (Fig. 4B). Similar to the S. rectivirgula–exposed PKD1-sufficient mice, the major cell type recovered from the airways and lung tissues isolated from PKD1-insufficient mice was neutrophils. Although they did not reach statistical significance, frequencies of neutrophils in BAL cells (at 6 h, p = 0.0903; at 24 h, p = 0.0705) and ILCs (p = 0.0856) from S. rectivirgula–exposed PKD1-insufficient mice were slightly lower compared with those of S. rectivirgula–exposed PKD1-insufficient mice. The numbers of neutrophils recovered from the BALF or lung tissue from PKD1-insufficient mice were ∼20 and 28%, respectively, of those in S. rectivirgula–exposed PKD1-sufficient mice. Of note, S. rectivirgula–mediated neutrophil influx into the lungs of PKD1fl/fl mice treated with 4-HT were comparable to those in vehicle-treated PKD1fl/fl mice, indicating that the observed reduction in alveolitis in PKD1-insufficient mice is not due to 4-HT treatment but due to the PKD1 insufficiency (Fig. 3C). Taken together, these results demonstrate that PKD1 plays a significant role in S. rectivirgula–induced neutrophilic alveolitis.
PKD1 is necessary for neutrophil recruitment into the airways and interstitial lung spaces following S. rectivirgula exposure.
(A) PKD1fl/fl-R26Cre/ERT2 mice were treated with vehicle (WT) or 4-HT (PKD1KO) six times by i.p. injection, as described in Materials and Methods. Within 1 wk after the last 4-HT treatment, mice were exposed intranasally to saline or S. rectivirgula for 6 h or 24 h. BAL was performed, and the BAL cells were recovered. Interstitial lung tissue cells (ILCs) were isolated from the whole lung from each mouse. Cells were counted to determine the degree of alveolitis using trypan blue exclusion. Total neutrophil cell count in BAL cells was derived by staining BAL cells with Abs to Gr-1, CD11b, and/or F4/80 followed by flow cytometry as described in Materials and Methods. Neutrophils were identified as CD11b+Gr1+F4/80−, and the frequency of neutrophils is expressed as the percentage of BAL cells. Total neutrophil cell count in ILCs was derived by staining ILCs with Abs to CD45, Gr-1, CD11b, and/or F4/80 followed by flow cytometry as described in Materials and Methods. Neutrophils in ILCs were identified as CD45+CD11b+Gr1+F4/80−, and the frequency of neutrophils is expressed as the percentage of ILCs. The number of mice used for each group is as follows: for 6 h S. rectivirgula challenge with WT-saline, n = 2; WT–S. rectivirgula, n = 4; PKD1KO–S. rectivirgula, n = 4; for 24 h S. rectivirgula challenge with WT-saline, n = 5; WT–S. rectivirgula, n = 4; PKD1KO–S. rectivirgula, n = 9. Data represent the mean cell number ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (B) PKD1fl/fl-R26Cre/ERT2 mice (n = 3/group) were treated with vehicle (WT) or 4-HT (PKD1KO) six times by i.p. injection, as described in Materials and Methods. Within 1 wk after the last 4-HT treatment, mice were exposed intranasally to saline or S. rectivirgula for 18 h. Representative H&E staining of the left lung lobe sections from mice exposed to the indicated stimuli are shown. The Aperio ScanScope XT slide scanner system was used to capture whole-slide digital images. The images presented ×3 original magnification (scale bars, 1000 μm) and ×60 original magnification (scale bars, 60 µm). All experiments were done two to three times with similar results. SR, S. rectivirgula.
Leukocyte infiltration into the airways and interstitial lung space after exposure to S. rectivirgula is a recurring phenotype for individuals with HP (2, 56, 57). To determine whether PKD1 also contribute to the leukocyte influx following repeated intranasal exposures to S. rectivirgula, PKD1-sufficient mice and PKD1-insufficient mice were exposed to S. rectivirgula three times per week for 5 wk, as indicated in (Fig. 5A, and BAL was performed 48 h after the last S. rectivirgula exposure. As shown in (Fig. 5B, PKD1 insufficiency resulted in significant suppression of alveolitis after repeated S. rectivirgula exposures. The numbers of PMNs (CD11b+Gr1+), AMs (CD11c+F4/80+), and CD4+ T cells were significantly reduced in the airway of PKD1-insufficient mice repeatedly exposed to S. rectivirgula for 5 wk compared with those in the PKD1-sufficient mice. However, the numbers of CD8+ T cells were not statistically different in the airway of PKD1-insufficient mice repeatedly exposed to S. rectivirgula for 5 wk compared with those in the PKD1-sufficient mice and in the saline-treated control mice. Cellular composition of alveolitis following a 5-wk exposure to S. rectivirgula was not different between PKD1-sufficient mice and PKD1-insufficient mice (Table II). The levels of surface expression of a costimulatory molecule CD86 and MHC-II on PMNs and AMs, as well as an early activation marker CD69 on CD4+ T cells and CD8+ T cells isolated from bronchial spaces of these mice, were also detected by flow cytometry. As shown in (Fig. 5C, levels of surface expression of CD86 on PMNs and AMs, as well as CD69 on CD4+ T cells and CD8+ T cells isolated from PKD1-insufficient mice, were comparable to those from PKD1-sufficient mice. There was slight, but significant, reduction in the levels of surface expression of MHC-II in PMNs (∼16% reduction in gMFI) and AMs (∼20% reduction in gMFI) isolated from airways of PKD1-insufficient mice compared with those from PKD1-sufficient mice. Our results suggest that PKD1 is one of major signaling contributors that lead the process of leukocyte infiltration into the bronchial space following repeated exposures to S. rectivirgula.
PKD1 insufficiency results in significant reduction in alveolitis and MHC-II surface expression on alveolar macrophages and polymorphonuclear neutrophils following repeated exposures to S. rectivirgula.
(A) Intranasal administration scheme for repeated S. rectivirgula exposures. (B and C) PKD1fl/fl-R26Cre/ERT2 mice were treated with vehicle (WT) or 4-HT (PKD1KO) six times by i.p. injection, as described in Materials and Methods. Within 1 wk after the last 4-HT treatment, mice were exposed intranasally to saline or S. rectivirgula three times per week for 5 wk and analyzed 48 h after the last S. rectivirgula exposure. BAL was performed, and the BAL cells were recovered. (B) BAL cells were counted to determine the degree of alveolitis using trypan blue exclusion. Total cell counts for polymorphonuclear neutrophils (PMNs; CD11b+Gr1+), alveolar macrophages (AMs; CD11c+F4/80+), CD4+ T cells (CD4+βTCR+), and CD8+ T cells (CD8+βTCR+) were derived by staining BAL cells with fluorochrome-conjugated Abs followed by flow cytometry as described in Materials and Methods. Data represent the mean cell number ± SD (n = 3–6 mice/group). Significance was determined by one-way ANOVA with a Tukey post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (C) BAL cells were stained with fluorochrome-conjugated Abs and then analyzed by flow cytometry and FlowJo flow software as described in Materials and Methods. Levels of surface expression of CD86 and MHC-II on PMNs and AMs and CD69 on CD4+ T cells and CD8+ T cells were determined by geometric mean fluorescent intensity (gMFI) of each marker in the indicated cell population. Data represent the mean gMFI ± SD (n = 4 mice/group). Significance was determined by two-tailed Student t test. *p < 0.05). SR, S. rectivirgula.
Table II.
Cellular composition of alveolitis following 5-wk exposure of WT and PKD1KO mice to S. rectivirgula
PMNs (%) .
AMs (%) .
CD4+βTCR+ (%) .
WT 57.16 ± 13.32 5.74 ± 1.27 10.88 ± 3.49 4.36 ± 2.47
PKD1KO 61.12 ± 13.60 6.13 ± 4.01 9.10 ± 2.97 4.29 ± 2.16
Values are given as mean ± SD of each group. PKD1-sufficient mice (WT; n = 3) and PKD1-insufficient mice (PKD1KO; n = 6) were exposed to S. rectivirgula (100 μg) three times per week for 5 wk and analyzed 48 h after the last exposure. The frequencies of polymorphonuclear cells (PMNs; CD11b+Gr1+), alveolar macrophages (AMs; CD11c+F4/80+), CD4+ T cells (CD4+βTCR+), and CD8+ T cells (CD8+βTCR+) in BAL cells were measured by flow cytometry followed by analysis using FlowJo software as described in Materials and Methods and expressed as the percentage of BAL cells.
Repeated exposures to HP-inciting Ags lead to the formation of granulomas, a focal aggregate of immune cells. To determine whether PKD1 contributes to the immune cell influx into the lungs following repeated exposures to S. rectivirgula, ILCs were isolated from PKD1-sufficient mice and PKD1-insufficient mice exposed to S. rectivirgula three times per week for 5 wk, and flow cytometric analysis was performed 48 h after the last S. rectivirgula exposure. As shown in (Fig. 6A, compared with the saline-treated control mice, PKD1-sufficient mice repeatedly exposed to S. rectivirgula for 5 wk showed significant increases in the percentage of CD11b+CD11c+ cells, CD11c+CD11b− cells, and CD4+βTCR+ cells, whereas they showed significant decreases in the percentage of CD8+βTCR+ cells, and no significant change in the percentages of CD11b+Gr1+ cells (p = 0.2515) and CD11b+F4/80+ cells. The frequencies of CD11b+F4/80+ cells, CD11b+Gr1+ cells, CD11c+CD11b− cells, CD4+βTCR+ cells, and CD8+βTCR+ cells within the interstitial lung tissue were not different between PKD1-sufficient mice and PKD1-insufficient mice following repeated exposures to S. rectivirgula. However, the frequency of CD11b+CD11c+ cells was significantly lower in PKD1-insufficient mice than in PKD1-sufficient mice. We observed no difference on levels of MHC-II surface expression on CD11b+CD11c+ cells and CD11c+CD11b− cells between mice exposed to saline and mice repeatedly exposed to S. rectivirgula (Fig. 6B). In contrast, repeated exposures to S. rectivirgula led to significant increases in the levels of MHC-II surface expression on CD11b+F4/80+ cells and CD11b+Gr1+ cells in PKD1-sufficient mice. These increases in levels of MHC-II surface expression on CD11b+F4/80+ cells and CD11b+Gr1+ cells following repeated exposures to S. rectivirgula were almost completely (if not completely) ablated in PKD1-insufficient mice. Our results showed that PKD1 contributes to the increased frequency of CD11b+CD11c+ dendritic cells and increased surface expression of MHC-II on macrophages and PMNs following repeated exposures to S. rectivirgula. The surface expression levels of an early activation marker CD69 on CD4+ T cells and CD8+ T cells in the interstitial lung tissues were significantly increased following repeated exposures to S. rectivirgula in PKD1-sufficient mice compared with those in PKD1-sufficient mice exposed to saline. The levels of the surface expression of CD69 on CD4+ T cells (p = 0.2641) and CD8+ T cells (p = 0.3438) were not statistically different between PKD1-sufficient mice and PKD1-insufficient mice following repeated exposures to S. rectivirgula. To determine whether PKD1 contributes to the development of granulomas, we examined lung tissue sections from PKD1-sufficient mice and PKD1-insufficient mice that had been exposed to S. rectivirgula for 5 wk. As shown in (Fig. 5C, whereas mice exposed to saline showed normal lung architecture, PKD1-sufficient mice exposed to S. rectivirgula for 5 wk exhibited granuloma formation. The PKD1-insufficient mice exposed to S. rectivirgula for 5 wk showed substantially reduced granuloma formation compared with PKD1-sufficient mice. Collectively, these results demonstrate that PKD1 contributes to leukocyte infiltration in the lungs, increased expression of MHC-II on myeloid lineage cells, and subsequent granuloma formation following repeated exposures to S. rectivirgula.
Effects of PKD1 insufficiency on the cellular composition of ILCs and granuloma formation following repeated exposures to S. rectivirgula.
PKD1fl/fl-R26Cre/ERT2 mice were treated with vehicle (WT) or 4-HT (PKD1KO) six times by i.p. injection, as described in Materials and Methods. Within 1 wk after the last 4-HT treatment, mice were exposed intranasally to saline or S. rectivirgula three times per week for 5 wk and analyzed 48 h after the last S. rectivirgula exposure. (A and B) ILCs were isolated from the whole lung from each mouse. ILCs were stained with fluorochrome-conjugated Abs and then analyzed by flow cytometry and FlowJo flow software as described in Materials and Methods. (A) The frequency of the indicated cell population is expressed as percentage of ILCs. (B) Levels of surface expression of MHC-II and CD69 were determined by geometric mean fluorescence intensity (gMFI) of each marker in the indicated cell population. Data represent the mean ± SD (n = 3–7 mice/group). Significance was determined by one-way ANOVA with a Tukey post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001. (C) Representative H&E staining of the left lung lobe sections from mice exposed to the indicated stimuli are shown (n = 5 mice/group). Each column represents the lung collected from an individual mouse. The Aperio ScanScope XT slide scanner system was used to capture whole-slide digital images. The images presented are ×4 original magnification (scale bars, 600 μm) and ×20 original magnification (scale bars, 200 µm). SR, S. rectivirgula.
Th1- and Th17-associated cytokines and chemokines are critical to the development and severity of HP (58–60). The regulatory cytokine IL-10 has been known to modulate inflammation and granuloma formation in HP (3). The Th2 cytokine IL-4 inhibits Th1 and Th17 differentiation and is known to play a suppressive role in HP (61–64). To investigate whether PKD1 contributes to the expression of any Th1/Th2/Th17-associated cytokines and chemokines following repeated exposures to S. rectivirgula for 5 wk, expression levels of the several selected cytokines and chemokines were assessed either by ELISA (IFN-γ, TNFα, IL-17A, IL-21, and IL-23) or a multiplex sandwich assay (IL-1β, IL-2, IL-4, IL-6, and IL-10). As shown in (Fig. 7A, compared with the saline-exposed PKD1-sufficient mice or S. rectivirgula–exposed PKD1-insufficient mice, slightly increased levels of IFN-γ in BALF obtained from PKD1-sufficient mice 48 h after the last S. rectivirgula exposure were detected (saline versus S. rectivirgula–treated PKD1-sufficient mice, p = 0.1102; S. rectivirgula–treated PKD1-sufficient mice versus S. rectivirgula–treated PKD1-insufficient mice, p = 0.1352). The levels of IFN-γ in BALF obtained from PKD1-insufficient mice exposed to S. rectivirgula repeatedly for 5 wk were not different from those from saline-exposed control mice (p = 0.9385). The levels of IL-17A in BALF obtained from PKD1-sufficient mice following repeated exposures to S. rectivirgula were significantly increased compared with those from saline-exposed mice. The levels of IL-17A in BALF obtained from PKD1-insufficient mice repeatedly exposed to S. rectivirgula were significantly reduced compared with those from S. rectivirgula–exposed PKD1-sufficient mice but were not different from those of saline-exposed control mice. Similar to the protein levels in BALF, mRNA levels of IFN-γ and IL-17A in the lungs of PKD1-sufficient mice exposed to S. rectivirgula for 5 wk were significantly increased compared with those of PKD1-sufficient mice exposed to saline for 5 wk (Fig. 7B). The levels of IFN-γ expression in the lungs of PKD1-insufficient mice repeatedly exposed to S. rectivirgula were not significantly different from those of PKD1-sufficient mice exposed to S. rectivirgula (S. rectivirgula–treated PKD1-sufficient mice versus S. rectivirgula–treated PKD1-insufficient mice, p = 0.2591). The levels of IL-17A expression in the lungs from PKD1-insufficient mice repeatedly exposed to S. rectivirgula were significantly reduced compared with those of S. rectivirgula–exposed PKD1-sufficient mice but were not different from those of saline-exposed control mice. We were not able to detect cytokines TNF-α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-21, and IL-23 above the assay detection sensitivity level in BALF obtained from PKD1-sufficient mice and PKD1-insufficient mice following repeated exposures to saline or S. rectivirgula (data not shown). IL-17RA mRNA levels in the lung were not different between saline-exposed control mice, S. rectivirgula–exposed PKD1-sufficient mice, and S. rectivirgula–exposed PKD1-insufficient mice (data not shown). Our results indicate that PKD1 plays a substantial role in the expression of IL-17A in mouse lungs following repeated exposures to S. rectivirgula.
PKD1 contributes to the increased expression of Th1/Th17-related cytokines and chemokines in lungs following repeated exposures to S. rectivirgula.
PKD1fl/fl-R26Cre/ERT2 mice were treated with vehicle (WT) or 4-HT (PKD1KO) six times by i.p. injection, as described in Materials and Methods. Within 1 wk after the last 4-HT treatment, mice (n = 3–6 mice/group) were exposed intranasally to saline or S. rectivirgula three times per week for 5 wk and analyzed 48 h after the last S. rectivirgula exposure. (A) Bronchoalveolar lavage (BAL) was performed. Levels of IFN-γ and IL-17A were detected by either ELISA. Data represent the mean concentration (pg/ml) ± SD. (B and C) Total RNA was purified from lung lobes isolated from each individual mouse and reverse transcribed. mRNA levels of the indicated genes were analyzed in duplicates by quantitative RT-PCR using a TaqMan Assay (for IL-17A, CXCL9, and CCL20) or SYBR Green Assay (for IFN-γ). The data on genes that were differentially expressed were normalized to the expression of the housekeeping gene, actin. Fold change comparing S. rectivirgula–treated exposed PKD1-sufficient mice and S. rectivirgula–treated PKD1-defficient mice to control PKD1-sufficient mice exposed to saline were calculated by comparative quantification algorithm-ΔΔCt method (fold difference = 2−ΔΔCt). Data represent the mean (fold) ± SD. Significance was determined by one-way ANOVA with a Tukey post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001 SR, S. rectivirgula.
By binding to specific receptors expressed on the surface of leukocytes, chemokines direct leukocyte movement. Chemokines CXCL9 (MIG), CXCL11 (I-TAC), and CCL20 (MIP-3α) are known to contribute to the Th1/Th17 polarization (65, 66). We analyzed whether expression of these chemokines in the lung following repeated exposure to S. rectivirgula is changed and whether PKD1 contributes to the S. rectivirgula–mediated changes in their expression. We found that the level of CXCL9 mRNA expression was significantly increased in the lungs of PKD1-sufficient mice following repeated exposure to S. rectivirgula (Fig. 7C). Compared to the expression level in PKD1-sufficient mice, expression levels of CXCL9 in the lung of PKD1-insufficient mice following repeated exposure to S. rectivirgula were significantly suppressed. The mRNA expression levels of CXCL11, CXCL12, CXCL13, and CCL20 in the lungs were not different between saline-exposed control mice, S. rectivirgula–exposed PKD1-sufficient mice, and S. rectivirgula–exposed PKD1-insufficient mice under our experimental condition (data not shown). Taken together, these results suggest that PKD1 contributes substantially to the expression of IL-17A and the Th1/Th17–associated chemokine CXCL9 following repeated exposures to S. rectivirgula.
The acute phase of HP is characterized by production of proinflammatory cytokines and a neutrophilic influx into the lung within hours of Ag exposure (2, 56, 57). Our previous studies using the S. rectivirgula mouse model of HP demonstrated that these responses are largely dependent on the TLR2-, TLR9-, and TLR/IL-1R–signaling adaptor MyD88 (36, 38). Previously, we have demonstrated that S. rectivirgula induces activation of PKD1, but not PKD2 or PKD3, through a MyD88-dependent mechanism and that PKD1 is one of the key signaling intermediaries downstream of MyD88 that lead to the expression of proinflammatory cytokines and chemokines in several murine cell lines following exposure to S. rectivirgula (49). Treatment of mice with the pharmacological agent Gö6976, which inhibits all three PKD family members and PKCα, PKCβ, and checkpoint kinases 1 and 2 (50, 51), ablated S. rectivirgula–mediated induction of alveolitis, pointing to a possibility of the essential role of PKD1 in the pulmonary inflammatory response to S. rectivirgula (49). However, due to the lack of specificity of the inhibitor for PKD1, contributions of PKD1 to development of the acute pulmonary inflammatory response and HP following exposures to S. rectivirgula have not been revealed. To investigate the role of PKD1 in S. rectivirgula–induced pulmonary inflammation and the development of HP, we employed tamoxifen-inducible PKD1-insufficient mice. The results from the current study provide evidence that PKD1 contributes substantially to production of proinflammatory cytokines and chemokines, leukocyte infiltration into the lungs, and granuloma formation following exposures to S. rectivirgula.
Activation of PKD1 by S. rectivirgula and inhibition of S. rectivirgula–mediated MAPK activation, NF-κB activation, and subsequent proinflammatory cytokine and chemokine production in vitro and in vivo by inhibitors for PKD, such as Gö6976, strongly suggested the role of PKD1 in S. rectivirgula–induced cytokine and chemokine production (49). In agreement with these previous findings, lungs obtained from PKD1-insufficient mice 1 h after exposure to S. rectivirgula were deficient in phosphorylation of MAPKs (JNK, ERK, and p38) and reduced IκBα degradation (an indication of NF-κB activation), demonstrating that PKD1 plays an indispensable role in S. rectivirgula–induced activation of these signaling modulators that are involved in expression of numerous proinflammatory mediators. Our previous studies demonstrated that induction of TNF-α, IL-6, MCP-1, and MIP-2, but not IL-1 or KC, following exposure to S. rectivirgula was completely dependent on MyD88 and partially dependent on TLR2 and TLR9 (36, 38). Using synthetic TLR ligands, we also found previously that PKD1 is indispensable for MyD88-dependent expression of TNFα, IL-6, IL-10, IL-12, IP-10, MCP-1, CCL5, and CD86 but dispensable for MyD88-dependent expression of type I IFNs and TRIF-dependent expression of cytokines and chemokines (34, 35). Similar to these previous findings, PKD1 was required for the optimal mRNA expression and protein production of IL-6, MIP-2, MCP-1, TNF-α, and IL-12 in the lungs in response to S. rectivirgula exposure. Especially, PKD1 appeared to be critical for S. rectivirgula–induced IL-12 production. However, PKD1 was not necessary for IFN-β mRNA expression or IL-1α, IL-1β, or KC production. These results suggest that PKD1 is a critical component of the TLR/MyD88 signaling pathways leading to proinflammatory cytokine and chemokine production following S. rectivirgula stimulation. Our results also indicate the presence of PKD1-independent pathways mediating signals downstream of S. rectivirgula and pattern recognition receptors, including TLR2, TLR9, and others yet to be identified. Patients with HP express increased levels of TNF-α, IL-6, and IL-8 in the BALF (4, 7, 9, 67, 68). Although PKD1 activation is not the absolute requirement for proinflammatory response to S. rectivirgula, our results suggest that PKD1 significantly contributes to the S. rectivirgula–induced proinflammatory response in the lungs, and therapeutics targeting PKD1 can be a valuable option for ameliorating these early inflammatory events in patients with HP.
HP patients develop a neutrophilic alveolitis shortly after Ag exposure. The neutrophils isolated from the lungs of patients with the chronic form of HP express high levels of gelatinase B and collagenase-2 that correlated with fibrosis (69, 70). Inhibition of neutrophil recruitment in a mouse model of HP resulted in a decrease in inflammation, supporting the idea that neutrophils play a role in pathogenesis (38). The in vitro studies demonstrating the importance of PKD1 in induction of cytokines and chemokines that contribute to neutrophil recruitment such as TNF-α, MIP-2, and MCP-1 suggest that PKD1 may play a critical role in neutrophil recruitment during HP. Our previous studies using the PKD- and PKC-specific inhibitor Gö6976 also pointed to a role for the PKD family in neutrophil recruitment into the lungs following exposure to S. rectivirgula (49). One-time exposure of PKD1-insufficient mice to S. rectivirgula resulted in significantly decreased alveolitis and a decrease in immune cells recruited into the interstitial lung tissue compared with PKD1-sufficient mice. The decrease in immune cells in the lungs and airspaces was attributed to a decrease in neutrophils. This was confirmed in H&E-stained lung sections, which demonstrated that the lungs of PKD1-insufficient mice are similar to those of saline-exposed mice. The reduction in neutrophils in the lungs correlated with a reduction in IL-6, IL-12, TNF-α, MCP-1, and MIP-2 in BALFs and lung tissues. The reduced production of these cytokines and chemokines is likely responsible for the low level of neutrophil recruitment that was observed in the PKD1-insufficent mice. Taken together, our findings demonstrate the importance of PKD1 in acute inflammation in this model.
The initial proinflammatory cytokine/chemokine environment and neutrophil infiltration might affect the outcome of disease development and progress. Our findings in PKD inhibitor–treated mice (49) and PKD1-insufficient mice after single exposure to S. rectivirgula raise the possibility that PKD1 may play a pivotal regulatory role in the pathogenesis of HP caused by repeated pulmonary exposures to S. rectivirgula. In agreement with this prediction, repeated exposures of PKD1-insufficient mice to S. rectivirgula resulted in significantly decreased alveolitis and a substantial reduction in granuloma formation compared with PKD1-sufficient mice. In our experimental setting, repeated exposures of PKD1-sufficient mice to S. rectivirgula for a 5-wk period resulted in significantly increased frequencies of dendritic cells (CD11b+CD11c+ cells and CD11b−CD11c+ cells) and CD4+ T cells in interstitial lung spaces. However, the increased frequency of monocyte-derived dendritic cells (CD11b+CD11c+ cells) was not observed in PKD1-insufficient mice exposed to S. rectivirgula repeatedly for 5 wk. In addition, although it is not statistically significant, frequencies of CD11b−CD11c+ cells and CD4+ T cells in interstitial lung spaces of PKD1-insuffcient mice following repeated exposures to S. rectivirgula were slightly reduced compared with those in PKD1-sufficent mice. Although the mechanism by which PKD1 is involved in S. rectivirgula–induced influx of monocyte-derived dendritic cells into the lung is yet to be revealed, it may be related to reduced production of MCP-1, one of chemokines that can attract dendritic cells to the site of inflammation, in the PKD1-insufficient mice following S. rectivirgula exposures. These findings suggest that in addition to the initial neutrophil infiltrations, PKD1 might play a critical role in subsequent recruitment of monocyte-derived dendritic cells into the lungs following repeated exposures to S. rectivirgula, which will affect the process of Ag presentation and granuloma formation during the pathogenic course of S. rectivirgula–mediated HP.
MHC-II, the molecules that present extracellular Ag-derived peptides to CD4+ T cells, is constitutively expressed in APCs (dendritic cells and macrophages) (71, 72). Under certain disease conditions, certain non-APCs acquire MHC-II expression and Ag presentation capacity (73). Tissue-infiltrated blood monocytes acquire MHC-II expression progressively as they mature to macrophages (74, 75). Expression of MHC-II can be induced in mouse neutrophils (76). MHC-II–expressing neutrophils function as a professional APC specializing in driving generation of proinflammatory Th1 and Th17 effector cells (76). Our results show that repeated exposures to S. rectivirgula led to the increases in MHC-II surface expression on macrophages and PMNs in the lungs of PKD1-sufficient mice. These increases in MHC-II surface expression following repeated exposures to S. rectivirgula appear to be influenced by PKD1. MHC-II surface expression levels on macrophages and PMNs at 6 and 24 h following one-time exposure to S. rectivirgula were not different between PKD1-sufficient mice exposed to saline and PKD1-sufficient mice and PKD1-insufficient mice exposed to S. rectivirgula (data not shown), indicating that these newly infiltrated cells may acquire MHC-II expression and Ag presentation ability on the site during the periods while the host is repeatedly exposed to S. rectivirgula. It is yet to be delimitated how PKD1 is involved in regulation of MHC-II expression in macrophages and neutrophils following repeated exposures to S. rectivirgula. There are several possible mechanisms by which PKD1 regulates surface expression of MHC-II. PKD1 regulates the fission from the trans-Golgi network of cargos that are destined to the cell surface (77). Several TLR ligands, especially the TLR2 ligand, induces surface expression of MHC-II in porcine monocytes and monocyte-derived dendritic cells (78). Thus, it is possible that PKD1 activated by S. rectivirgula (presumably through TLR2) promotes transportation of MHC-II–containing cargo from trans-Golgi network to the cell surface. This possibility is warranted to be tested in the future. Inflammatory cytokine IFN-γ plays a critical role on maturation of newly infiltrated blood monocytes, which are MHC-II–negative cells, to functional APCs that express MHC-II on their surfaces (73). IFN-γ is also a potent inducer of MHC-II in both APCs and non-APCs, allowing them to acquire Ag presentation capacity (79). Thus, it is possible that PKD1 contributes to the expression of MHC-II by directly and/or indirectly involving IFN-γ expression in the lungs following exposure to S. rectivirgula. IFN-γ expression in the lungs of mice following repeated S. rectivirgula exposures is dependent on TLR9, one of the TLRs that use PKD1 as a key signaling intermediary (36). S. rectivirgula–induced expression of IFN-γ in MLE12 cells (a murine bronchial epithelial cell line) is dependent on PKD1 (49). S. rectivirgula–induced expression of IL-12, a cytokine that can induce expression of IFN-γ (80), in MPRO (promyelocytes differentiated to neutrophils) and AMJ2-C11 (AMs) is dependent on PKD1 (49). Our study also shows that PKD1 plays an indispensable role in IL-12 expression, a cytokine that is involved in MHC-II expression by upregulating IFN-γ expression (81), in mouse lungs following exposure to S. rectivirgula. Taken together, these findings support the possibility that PKD1 contributes to the increased expression of MHC-II on macrophages and neutrophils following repeated exposures to S. rectivirgula by directly and/or indirectly involving IFN-γ production. It will be interesting to further pursue whether and how PKD1 plays a role in S. rectivirgula–induced IFN-γ expression and whether IFN-γ is responsible for increased surface expression of MHC-II on macrophages and neutrophils following repeated exposures to S. rectivirgula. Inducible MHC-II expression is often associated with inflammation. The TLR/IL-1R signaling adaptor MyD88 is necessary for neutrophil recruitment and pulmonary inflammation following exposures to S. rectivirgula (38). Although it is not complete abolition similar to what was observed in MyD88-deficient mice, PKD1 contributes significantly to neutrophil infiltration into the lungs and pulmonary inflammation following exposure to S. rectivirgula. Thus, we cannot rule out the possibility that the increased MHC-II surface expression on PMNs and macrophages in the lungs of mice following repeated exposures to S. rectivirgula could be the result of increased inflammation that was significantly inhibited in the absence of PKD1.
Th1- and Th17-associated cytokines and chemokines are critical to development of HP and the disease severity (58–60). TLR2, TLR6, and TLR9 contribute significantly to expression of IL-17A and Th17 cell generation following repeated exposure to S. rectivirgula (36, 82). Similar to these previous findings, our study demonstrates that PKD1, which can act as a MyD88-dependent TLR/IL-1R signaling modulator, also contributes significantly to expression of IFN-γ, IL-17A, and Th1/Th17-polarizing chemokine CXCL9 following repeated exposures to S. rectivirgula. No significant change in CXCL11 and CCL20 expression in lungs following repeated exposures to S. rectivirgula was detected under our experimental condition. Unlike CXCL9/CXCR3 binding, CXCL11/CXCR3 binding induces Foxp3-negative IL-10hi regulatory T cells (Tr1) and IL-4hi (Th2) cells (83). IL-10 and IL-4 were also not detected in BALF obtained from PKD1-sufficient mice and PKD1-insufficient mice following repeated exposures to S. rectivirgula. These results imply that the reduced Th1/Th17-associated responses observed in PKD1-insufficient mice is not skewing S. rectivirgula–induced Th1/Th17 immune response toward Th2 activity.
In summary, using PKD1-insufficient mice, we found that PKD1 contributes substantially for S. rectivirgula–induced production of proinflammatory cytokines TNF-α, IL-6, IL-12, IFN-γ, and IL-17A and chemokines MIP-2, MCP-1, and CXCL9 in the lung in response to S. rectivirgula. PKD1 contributes to the development of an alveolitis, increased expression of MHC-II in myeloid lineage cells, and granuloma formation following repeated exposures to S. rectivirgula. Taken together, our findings imply that PKD1 activation may play an essential role in pulmonary proinflammatory responses and HP caused by S. rectivirgula and that inhibiting PKD1 activity or expression could be a valuable adjunctive therapeutic option for controlling HP at an early stage.
We thank Dr. E. Olson (University of Texas Southwestern Medical Center, Dallas, TX) for providing PKD1fl/fl mice. The Flow Cytometry Facility in Research Service at the Memphis V.A. Medical Center is also gratefully acknowledged.
A.-K.Y. was supported by National Institutes of Health Grants AR064723 and AR069010. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health or The University of Tennessee Health Science Center.
T.W.Y., E.A.F., and A.-K.Y. designed the study; T.W.Y., E.A.F., J.D.S., S.L., Y.-I.K., and C.Z. performed experiments and acquired the data; T.W.Y., E.A.F., J.D.S., S.L., Y.-I.K., and A.-K.Y. analyzed and interpreted the data; T.W.Y., E.A.F., S.L., and A.-K.Y. wrote the manuscript. All authors proofread and gave their approval for the final submitted version.
alveolar macrophage
bronchoalveolar lavage
BAL fluid
gMFI
geometric mean fluorescence intensity
hypersensitivity pneumonitis
4-HT
4-hydroxytamoxifen
interstitial lung cell
IL-1R–associated kinase
keratinocyte chemoattractant
LPS-induced CXC chemokine
MHC-II
MHC class II
PKC
protein kinase C
protein kinase D
PKD1KO
PKD1 knockout
PMN
polymorphonuclear neutrophil
R26Cre/ERT2
Gt(ROSA)26Sortm1(cre/ERT2)Tyj/J
TRAF6
TNFR-associated factor 6
UTHSC
The University of Tennessee Health Science Center
wild-type
Salvaggio
deShazo
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Wakeham
Itie
Severe impairment of interleukin-1 and Toll-like receptor signalling in mice lacking IRAK-4.
Galán
Janeway
IRAK-M is a negative regulator of Toll-like receptor signaling.
Yeh
The role of interleukin 1 receptor-associated kinase-4 (IRAK-4) kinase activity in IRAK-4-mediated signaling.
Upadhyay
B. K.
Group B streptococci induce proinflammatory responses via a protein kinase D1-dependent pathway.
Häcker
Vabulas
Immune cell activation by bacterial CpG-DNA through myeloid differentiation marker 88 and tumor necrosis factor receptor-associated factor (TRAF)6.
Akkaraju
Z. J.
TAK1 is a ubiquitin-dependent kinase of MKK and IKK.
Convergence of CpG DNA- and BCR-mediated signals at the c-Jun N-terminal kinase and NF-κB activation pathways: regulation by mitogen-activated protein kinases.
Role of mitogen-activated protein kinases in CpG DNA-mediated IL-10 and IL-12 production: central role of extracellular signal-regulated kinase in the negative feedback loop of the CpG DNA-mediated Th1 response.
Protein kinase D1 is essential for the proinflammatory response induced by hypersensitivity pneumonitis-causing thermophilic actinomycetes Saccharopolyspora rectivirgula.
Dieterich
Rennecke
Kittstein
Inhibition of protein kinase C μ by various inhibitors. Differentiation from protein kinase c isoenzymes.
Kohn
Yoo
The protein kinase C inhibitor Gö6976 is a potent inhibitor of DNA damage-induced S and G2 cell cycle checkpoints.
Fielitz
Bassel-Duby
Requirement of protein kinase D1 for pathological cardiac remodeling.
CpG DNA induces self and cross-hyporesponsiveness of RAW264.7 cells in response to CpG DNA and lipopolysaccharide: alterations in IL-1 receptor-associated kinase expression.
IFN-γ production by innate immune cells is sufficient for development of hypersensitivity pneumonitis.
Chemokine production during hypersensitivity pneumonitis.
Trentin
Facco
New aspects of hypersensitivity pneumonitis.
Curr. Opin. Pulm. Med.
Hypersensitivity pneumonitis: current concepts and future questions.
T-bet controls severity of hypersensitivity pneumonitis.
J. Inflamm. (Lond.)
Hogaboam
Interleukin-17-mediated immunopathogenesis in experimental hypersensitivity pneumonitis.
Simonian
Diaz del Valle
W. K.
Fontenot
Th17-polarized immune response in a murine model of hypersensitivity pneumonitis and lung fibrosis.
Reciprocal modulation between TH17 and other helper T cell lineages.
J. Cell. Physiol.
Bottomly
Induction of Th1 and Th2 CD4+ T cell responses: the alternative approaches.
Hwang
IL-4-deficient mice aggravate hypersensitivity pneumonitis.
Immune Netw.
IL-4-secreting NKT cells prevent hypersensitivity pneumonitis by suppressing IFN-γ-producing neutrophils.
Syrovets
Simmet
Recruitment of CCR6-expressing Th17 cells by CCL20 secreted from plasmin-stimulated macrophages.
Acta Biochim. Biophys. Sin. (Shanghai)
Tokunaga
Naseem
McSkane
CXCL9, CXCL10, CXCL11/CXCR3 axis for immune activation—a target for novel cancer therapy.
McClanahan
Zlotnik
Selman
CCL18/DC-CK-1/PARC up-regulation in hypersensitivity pneumonitis.
Bedard
A study of monokine release and natural killer activity in the bronchoalveolar lavage of subjects with farmer's lung.
Am. Rev. Respir. Dis.
Haslam
Z. S.
Primett
Newman-Taylor
Turner-Warwick
Mast cells, atypical lymphocytes, and neutrophils in bronchoalveolar lavage in extrinsic allergic alveolitis. Comparison with other interstitial lung diseases.
Gaxiola
Segura-Valdez
Mejía
Increase of lung neutrophils in hypersensitivity pneumonitis is associated with lung fibrosis.
Lipski
Dewispelaere
Caspers
Defrance
Bruyns
Willermain
MHC class II expression and potential antigen-presenting cells in the retina during experimental autoimmune uveitis.
LeibundGut-Landmann
Waldburger
Regulation of MHC class II gene expression by the class II transactivator.
Furuta
The ins and outs of MHC class II-mediated antigen processing and presentation.
Bravo-Blas
Perdiguero
Geissmann
Malissen
Mowat
Constant replenishment from circulating monocytes maintains the macrophage pool in the intestine of adult mice. [Published erratum appears in 2014 Nat. Immunol. 15: 1090.]
Jakubzick
Monocyte differentiation and antigen-presenting functions.
Abi Abdallah
B. A.
Denkers
Mouse neutrophils are professional antigen-presenting cells programmed to instruct Th1 and Th17 T-cell differentiation.
Liljedahl
Colanzi
Protein kinase D regulates the fission of cell surface destined transport carriers from the trans-Golgi network.
B. N.
Toll-like receptor, MHC II, B7 and cytokine expression by porcine monocytes and monocyte-derived dendritic cells in response to microbial pathogen-associated molecular patterns.
Vet. Immunol. Immunopathol.
Korman
Kappes
Fiers
Ault
Gimbrone
Strominger
Pober
Immune interferon activates multiple class II major histocompatibility complex genes and the associated invariant chain gene in human endothelial cells and dermal fibroblasts.
Ethuin
Gérard
Boutten
Gougereot-Pocidalo
Chollet-Martin
Human neutrophils produce interferon gamma upon stimulation by interleukin-12.
Schnyder
LeHir
Shakhov
Woerly
Aguet
Ryffel
Role of interferon-gamma in interleukin 12-induced pathology in mice.
Toll-like receptor 6 drives interleukin-17A expression during experimental hypersensitivity pneumonitis.
Wildbaum
Salzman
Thelen
Barsheshet
CXCL11-dependent induction of FOXP3-negative regulatory T cells suppresses autoimmune encephalomyelitis. [Published erratum appears in 2018 J. Clin. Invest. 128: 1200–1201.]


text:
Q: Libraries used in Android I've downloaded the source from https://android.googlesource.com and I now want know if I can compile the libraries(bionic) and Dalvik VM using a cross compiler? If yes, how can I do that?

A: All the dependency is given on http://source.android.com/source/download.html as follows:

$ sudo add-apt-repository "deb http://archive.canonical.com/ lucid
  partner" $ sudo add-apt-repository "deb-src
  http://archive.canonical.com/ubuntu lucid partner" $ sudo apt-get
  update $ sudo apt-get install sun-java6-jdk $ sudo
  update-java-alternatives -s java-6-sun $ sudo apt-get install
  git-core gnupg flex bison gperf build-essential zip curl zlib1g-dev
  gcc-multilib g++-multilib libc6-dev-i386 lib32ncurses5-dev ia32-libs
  x11proto-core-dev libx11-dev lib32readline5-dev lib32z-dev

redpajama_set_name: RedPajamaStackExchange


text:
Landslide Hazards
Seattle, WA Landslide Information
More Information on Seattle's Project Impact.
House in the Magnolia Bluffs area overlooking Puget Sound that was struck by a rapidly-moving landslide. The flow deposited material in the garage (upper level) and collapsed the front wall of the living quarters (lower level).
When wet winter weather sets in, many residents of the Puget Sound area begin to worry about landslides. Landslides that occur on bluffs and hillsides of communities surrounding Puget Sound, Washington, pose a serious hazard to people, property, utilities, transportation, and businesses. After major storms caused hundreds of destructive landslides during the winter of 1996-97, the USGS began studies of landslides in the Seattle area. Subsequently, at the beginning of Seattle's Project Impact in February 1998, the USGS and Seattle agreed to collaborate on producing state of the art landslide and seismic hazard maps for the city. The USGS recently completed its suite of landslide maps for Seattle and developed methods that can be used to forecast the occurrence of landslides.
The recent USGS landslide hazards analyses have benefited from several allied efforts, including the Seattle-sponsored compilation of its century-long landslide database, by its consultant, Shannon and Wilson, Inc.(see OFR-00-303 - Preliminary map showing landslide densities, mean recurrence intervals, and exceedance probabilities as determined from historic records, Seattle, Washington), acquisition of high-resolution topographic data by the Puget Sound LIDAR Consortium, and detailed geologic mapping by the Pacific Northwest Geologic Mapping & Urban Hazards. Landslide monitoring in cooperation with the Burlington Northern Santa Fe Railway, the Dominican Reflection Center, of Woodway, and the Washington Department of Transportation has contributed to an understanding of the action of precipitation in causing shallow landslides as well as deep-seated landslides.
Landslide on the flank of a small drainage in northwest Seattle. Foundation of house on right edge of photograph and decks of neighboring houses were undermined.
Back to Seattle, WA Area Monitoring
Status - Active


text:
Q: cURL returns Empty reply from server I am using following command but in return I am getting "curl: (52) Empty reply from server". Please see the code and advise what change it requires.
curl -k --user "USERNAME:PASSWORD" --request POST --header "Content-type: text/xml" --header "SOAPAction:http://EXAMPLE.COM/b2b/2014/02/IB2BService/SubmitMessage" --data @actual.xml https://EXAMPLE.COM/B2BGatewayService/B2BGatewayService.svc

Any suggestion is highly appreciable.