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. Author manuscript; available in PMC: 2015 Nov 15.
Published in final edited form as: J Immunol. 2015 Apr 6;194(10):4924–4930. doi: 10.4049/jimmunol.1401362

CCL7 and IRF7 mediate hallmark inflammatory and interferon responses following rhinovirus 1B infection

CCL7 and IRF-7 in the host response to rhinovirus

Jason Girkin *,†, Luke Hatchwell *,†, Paul Foster †, Sebastian L Johnston ‡, Nathan Bartlett ‡, Adam Collison *,†, Joerg Mattes *,†,§
PMCID: PMC4417644  EMSID: EMS62668  PMID: 25847975

Abstract

Rhinovirus infections are common and have the potential to exacerbate asthma. We have determined the lung transcriptome in RV strain 1B (RV1B) infected naïve BALB/c mice (non-allergic) and identified CCL7 and IRF7 amongst the most upregulated mRNA transcripts in the lung. To investigate their roles we employed anti-CCL7 antibodies and an IRF7-targeting small interfering RNA in vivo. Neutralising CCL7 or inhibiting IRF7 limited neutrophil and macrophage influx and IFN responses in non-allergic mice. Neutralising CCL7 also reduced activation of NF-κB p65 and p50 subunits, as well as airways hyperreactivity (AHR) in non-allergic mice. However, neither NF-κB subunit activation nor AHR were abolished with infection of allergic mice after neutralising CCL7, despite a reduction in the number of neutrophils, macrophages and eosinophils. IRF7 siRNA primarily suppressed IFN-α and -β levels during infection of allergic mice.

Our data highlight a pivotal role of CCL7 and IRF7 in RV-induced inflammation and IFN responses and link NF-κB signalling to the development of AHR.

Introduction

Rhinovirus (RV) infections are the most common factor associated with exacerbations of asthma(1, 2). Exacerbations of asthma induced by viral infections are difficult to treat and are a major cause of asthma morbidity, often associated with a loss of disease control by provocation of airways hyperreactivity (AHR) and lung inflammation(3, 4). As such, much research has been directed towards identifying the underlying mechanisms of RV-induced asthma exacerbations with the hope of developing novel therapeutics.

Normally RV infections induce a rapid production of IFN-α and IFN-β along with the recruitment of neutrophils following detection of RV by epithelial cells and macrophages(5-7). Robust IFN responses are capable of limiting inflammation and reducing the duration, severity and spread of viral infection(8). Many asthma sufferers generally exhibit elevated eosinophil and neutrophil recruitment upon RV infection and a diminished production of IFN-α and -β. Therefore, it is important to investigate the mechanisms of IFN production and general, antiviral and inflammatory responses. It is also of pivotal importance to study the chemokines released upon infection, due to their ability to induce leukocyte trafficking and their potential to control the intensity of an immune response(9).

In this study, we infected BALB/c mice with minor strain RV1B and conducted an array on RNA extracted from blunt dissected airway tissue. The most significantly upregulated genes following RV infection included the chemokine (C-C motif) ligand 7 (CCL7, also known as monocyte chemotactic protein -3 or MCP-3) and interferon regulatory factor -7 (IRF-7). This paper shows a functional role of CCL7 in the chemotaxis of neutrophils, macrophages and eosinophils in RV-induced exacerbation of allergic airways disease (AAD) in vivo and an important role of IRF-7 in promoting type 1 IFN responses to RV infection.

Materials and Methods

Animals

All experiments were approved by the Animal Care and Ethics Committee of the University of Newcastle. 6-8 week old male BALB/c mice obtained from the Specific Pathogen Free Facility of the University of Newcastle, were housed within approved containment facilities in Bioresources at the Hunter Medical Research Institute (Newcastle, Australia) with ad libitum access to food and water under a 12 hr light and dark cycle. All experiments were conducted in accordance with the ARRIVE guidelines.

Administration of CCL7 antibody and IRF7 siRNA

24 hours before inoculation with minor group RV strain 1B (RV), mice were intraperitoneally administered with 0.8mg/kg (20μg/200μl) anti-CCL7 antibody (anti-CCL7 – R&D Systems) or a species-matched isotype control antibody. In other experiments, mice were intranasally administered with 3.75nmol of an IRF-7-targetted siRNA (IRF7 siRNA) intranasally (3.75nmol/25μl) or nonsense control siRNA 24hrs before innoculation with RV. Both siRNAs were obtained from Dharmacon.

Induction of allergic airways disease and RV-induced exacerbation

Lyophilized preparation of milled house dust mites (HDM) containing Dermatophagoides pteronyssinus extract was obtained from Greer Laboratories (Lenoir, NC). Mice were sensitised and challenge to HDM followed by infection with RV, as previously reported(10, 11). Briefly, mice were treated intranasally under light isoflurane anaesthesia with HDM (50μg/50μl in sterile saline), daily over 3 days during sensitisation. Mice were then intranasally challenged 12 days later, with HDM (5μg/50μl) over 4 days to induce AAD. Nonsensitized mice (Saline) received sterile endotoxin-free saline only. Allergic (HDM) mice were intranasally infected with RV, (50μl containing 1×107 virions), or UV-inactivated RV, 24hrs after the last HDM challenge to exacerbate preexisting AAD. Mice were euthanized 24hrs after the RV infection at the peak of host response. 24hr post infection was chosen for our analyses as innate immune responses induced by RV1B in BALB/c mice peaks 24hrs post infection and RV infection is quickly resolved(12) by four days post-infection.

AHR measurement

AHR was measured as previously described(13-15). Briefly, mice were anaesthetised with ketamine-xylene combination (Illum) and total lung resistance and dynamic compliance was measured invasively (Buxco). Mice were mechanically ventilated and increased lung resistance to nebulised methacholine was expressed as percentage change from control (baseline).

Analysis of bronchoalveolar lavage

Bronchoalveolar lavage fluid (BALF) was collected by cannulating the trachea of euthanized mice and flushing the airways twice with 1ml of Hank’s Buffered Salt Solution. BALF was centrifuged at 800 × g for 10 min at 4°C. Pelleted cells were treated with Red Blood Cell Lysis buffer (12mM NaHCO3, 0.1mM EDTA, 155mM NH4Cl, pH 7.35) for 5 min and centrifuged as above. Cell pellets were resuspended in 100μl of HBSS and total number of viable cells determined by trypan blue exclusion in a haemocytometer. Following a cytospin, slides were stained with May-Grunwald-Giemsa, blinded and differential cell counts determined from a count of 200 cells per slide.

Histology

An excised lung lobe was fixed in 10% Neutral Buffered Formalin for 24 hours, then stored in 70% Ethanol. Carbol’s chromotrope-hematoxylin (CR) and Periodic acid-Schiff (PAS) stains of 5μm slices of fixed lungs were used for semiquantitative histopathologic scoring (range 0 to 10) as previously described(16). Scores are a sum of perivascular edema (0 = absent; 1 = mild to moderate, present in <25% of the perivascular spaces; 2 = moderate to severe, in 25%-75% of perivascular spaces; or 3, severe, present in >75% of perivascular spaces); perivascular/peribronchial acute inflammation (0 = absent; 1 = perivascular edematous inflammation with <5 neutrophils per high-power field; 2 = moderate perivascular inflammation, involving some peribronchial spaces, with >5 neutrophils per high power field; or 3 = severe, acute perivascular and peribronchial inflammation with numerous neutrophils encircling > 50% bronchioles); bronchiole goblet cell metaplasia (0 = absent; 1 = two or less bronchioles with <5 goblet cells; or 2 = large numbers of goblet cells present); and inflammation in alveolar spaces (0 = absent; 1 = present in <25% of alveolar spaces; or 2 = present in >25% of alveolar spaces). Photos representative of all mice in a group were taken from tissue sections using Imagepro Plus software and enhanced in Adobe Photoshop CS5.1 to reduce brightness (−30) and enhance contrast (+100)

Quantitative RT-PCR

Thoracic contents were extracted from euthanized mice and forceps were used to separate airways from the parenchyma by blunt dissection(17) resulting in the isolation of several generations of airway tissue. TRIzol® (Ambion, Carlsbad, USA) was used to extract airway mRNA and concentrations were determined by spectrophotometry (Nanodrop) for reverse transcription with BioScript™ (Bioline, Alexandria, Australia) to generate cDNA. Quantitative PCRs (qPCR) were performed according to manufacturers instructions with SYBR® Green (Invitrogen, Mulgrave, Australia) using the primer sequences in Supplementary Table 1. Copy numbers of target genes were obtained by referencing CTs to a standard curve of known concentration. All values were normalised to the endogenously expressed control gene, HPRT.

Flow cytometry

Mouse lung cells were mechanically extracted using GentleMACS tubes as per manufacturers instructions and stained with anti-CD11c-fluorescein isothiocyanate (FITC), anti-CD11b-peridinin-chlorophyll protein (PerCP) (Pharmingen), anti-MHCII-phycoerythrin (PE), and anti-F4/80-allophycocyanin (APC) (eBioscience) to determine mDC and macrophage numbers, or anti-mPDCA-1-APC to determine pDC numbers (Miltenyl). We determined numbers of positive cells by flow cytometry (FACS Canto, Becton Dickinson).

Quantification of lung chemokines, phosphorylated-ERK1 and active NFκB subunits

A single excised lung lobe from each mouse was snap frozen before homogenisation in buffers and protease inhibitors as recommended in manufacturers instructions. Levels of CCL7 (eBioscience), CCL11, CCL20, CXCL2 (R&D Systems), and phosphorylated-ERK1 were determined in clarified lung lysates by ELISA. Quantification of activate NFκB subunits was performed with a TransAM NFκB Transcription Factor Assay (Active Motif). All concentrations were normalised to lung lobe weight.

Statistical analyses

All figures were graphed in GraphPad Prism 6.0 (GraphPad Software, La Jolla, California), expressed as Mean±SEM. Student’s t-test or 2-way ANOVA were used as appropriate with an alpha value of 0.05 for all analyses. All significantly different results shown are a comparison to the isotype or nonsense control, unless otherwise stated.

Results

CCL7 is the most significantly upregulated gene induced by RV infection and Inhibition of CCL7 reduces inflammation and AHR

CCL7 is the most significantly upregulated gene at the peak of RV infection in vivo (www.ebi.ac.uk/arrayexpress, online repository number: E-MTAB-2826), confirmed by qPCR (Fig 1A) and ELISA (Fig 1B). Therefore, we administered a neutralising antibody against CCL7 (anti-CCL7) and subsequently infected BALB/c mice with RV. 24hr post infection, the peak of viral load and host response, mice were sacrificed for analyses. anti-CCL7 treatment did not affect chemokine (C-X-C motif) ligand 2 (CXCL2, also known as macrophage inflammatory protein 2-α or MIP2-α) release, demonstrating that the observed effects were specifically due to CCL7 inhibition (Fig 1B). anti-CCL7 suppressed AHR induced by RV (Fig 1C), concurrent with a decrease in neutrophil and macrophage numbers in BALF (Fig 1 D).

FIG 1. Neutralisation of CCL7 suppresses RV-induced AHR and lung inflammation.

FIG 1

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BALB/c mice infected with RV were assessed for CCL7 expression 24hr post infection. Mice were then treated with anti-CCL7 antibodies and subsequently infected with RV and sacrificed 24hrs post infection. (A) CCL7 mRNA upregulation validated by qPCR. (B) Levels of chemokines in whole lung homogenates as assessed by ELISA. (C) Total lung resistance presented as percentage change in response to methacholine (n=6-8). (D) Cell populations present in BALF as assessed by May-Grunwald-Giemsa staining. Results are Mean±SEM at 1 day post-infection (n=3-5 mice per group). *= P < 0.05, **= P < 0.01, ***= P <0.001 as compared to isotype control group.

CCL7 mediates pro-inflammatory transcription factors and antiviral responses

Intraperitoneal administration of anti-CCL7 24hrs prior to RV infection suppressed levels of the phosphorylated MAPK ERK1 and active NF-κB p65 and p50 (Fig 2A and B). anti-CCL7 treatment reduced expression of IRF-7 and IRF-5 mRNA, but not IRF-3 mRNA (Fig 2C) and dampened IFN-β release (Fig 2E). anti-CCL7 did not affect viral titre as determined by qPCR of positive strand RV1B RNA (Fig 2D). To explore the relationship between CCL7, IRF-7 and IFN-β, we assessed pDC and macrophage populations in the lung by flow cytometry. Flow cytometric analysis of pDCs revealed that anti-CCL7 did not reduce the number of pDCs recruited by RV (Fig 2F). However, alveolar macrophage numbers in lung tissue were reduced to baseline by anti-CCL7 (Fig 2F).

FIG 2. Neutralisation of CCL7 alters RV-induced MAPK, NF-kB activity and IFN production.

FIG 2

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BALB/c mice were infected with RV following administration of anti-CCL7 antibodies and sacrificed 24hrs post infection. (A) Levels of p-ERK1, (B) p65 and p50 in lung homogenates as assessed by ELISA. (C) mRNA expression of IRF-7, -5, -3 and (D) positive-strand RV1B RNA in blunt dissected airway tissue, assessed by qPCR. (E) IFN-α and -β in lung homogenates as assessed by ELISA. (F) Numbers of pDCs and alveolar macrophages present in mouse lungs determined by flow cytometry. All results are Mean±SEM at 1 day post-infection (n=3-6 mice per group). *, p < 0.05, **, p < 0.01. ***, p < 0.001. ****, p < 0.0001 as compared to isotype control group. #, p < 0.05 (one tailed) as compared to isotype control.

IRF-7 mediates type 1 IFN production and inflammation in response to RV Infection

To explore the role of IRF-7 upregulation during RV infection, we administered IRF-7 siRNA intranasally, 24hrs prior to RV infection. IRF-7 siRNA successfully suppressed IRF-7 mRNA expression at 24hr post infection without affecting other IRFs, such as IRF-5 or IRF-3 (Fig 3A). This resulted in complete suppression of IFN-α and IFN-β production, but –like inhibition of CCL7 and subsequent impaired IRF7 expression– had no effect on RV titre (Fig 3B and C). IRF-7 siRNA treatment reduced macrophage and neutrophil numbers in the BALF (Fig 3E), but this did not result in a significant reduction in AHR compared to RV infected mice that received nonsense siRNA control (Fig 3D). IRF-7 siRNA treatment did not affect CCL7 or CXCL2 production or reduce levels of p-ERK1 or active NF-κB subunits (Fig 3F, G and H).

FIG 3. Inhibiting IRF-7 suppresses RV-induced IFN responses and BALF cell recruitment.

FIG 3

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BALB/c mice infected with RV following administration of IRF-7 targeting siRNA and sacrificed 24hrs post infection. (A) mRNA expression of IRF-7, -5, and -3 in blunt dissected airway tissue, assessed by qPCR. (B) IFN-α and -β in lung homogenates as assessed by ELISA. (C) Copy numbers of positive-strand RV1B RNA from blunt disected airway tissue, assessed by qPCR. (D) Total lung resistance presented as percentage change in response to methacholine (n=6). (E) Cell populations present in the BALF, as assessed by May-Grunwald-Giemsa staining. (F) Levels of chemokines, (G) p-ERK-1 and (H) Active NF-κB p65 and p50 assessed by ELISA. All results are Mean±SEM at 1 day post infection (n=3-6 mice per group). *, p < 0.05, **, p < 0.01. ***, p < 0.001 as compared to nonsense control group.

CCL7 mediates airways inflammation during RV induced exacerbation of AAD

To investigate the role of CCL7 in RV induced exacerbations of AAD, we sensitised and challenged mice to HDM and on the day of final challenge, 24hrs prior to RV infection, mice received anti-CCL7 intraperitoneally. CCL7 was upregulated in mice treated with an isotype control antibody and was successfully neutralised in the lung after anti-CCL7 treatment (Fig 4A) and resolved after four days post-infection along with clearance of RV infection (Supplemental Figure 1). CCL7 inhibition also downregulated CCL20 (also known as macrophage inflammatory protein 3-α or MIP3-α), but not CXCL2 (Fig 4A) or CCL11 (also known as Eotaxin-1) (Fig 4B). BALF neutrophils, macrophages and eosinophils were reduced with anti-CCL7 (Fig 4C). However, anti-CCL7 treatment did not ameliorate AHR (Fig 4D). Furthermore, NF-κB p65 and p50 activity remained elevated in anti-CC7-treated allergic mice (Fig 4E). CCL7 neutralisation during RV infection of allergic did not affect IRF-7, type I IFN mRNA or RV1B copy numbers (Supplemental Figure 2).

FIG 4. Neutralisation of CCL7 suppresses host inflammatory responses during RV induced exacerbation of AAD.

FIG 4

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BALB/c mice were sensitized and challenged to HDM to induce allergic airways disease, then administered with anti-CCL7 antibodies and subsequently infected with RV to induce exacerbation. Mice were sacrificed 24hrs post infection for analyses. (A) Levels of chemokines in lung homogenates measured by ELISA. (B) mRNA expression of CCL11 in blunt dissected airway tissue. (C) Cell populations present in BALF, as assessed by May-Grunwald-Giemsa staining. (D) Total lung resistance presented as percentage change in response to methacholine (n=5-9). (E), Active NF-κB subunits in whole lung homogenates assessed by ELISA. All results are Mean±SEM at 1 dpi (n=3-6 mice per group). *, p < 0.05, **, p < 0.01. ***, p < 0.001. ****, p < 0.0001 as compared to isotype control group.

IRF-7 promotes IFN production during RV induced exacerbation of AAD

Next, we investigated the effects of IRF-7 inhibition in RV induced exacerbation of AAD. We sensitised and challenged mice to HDM and on the day of final challenge, 24hrs prior to RV infection, mice received IRF-7 siRNA intranasally. IRF-7 mRNA expression was significantly induced 24hr following RV infection and resolved after four days post-infection (Supplementary Figure 1). IRF-7 siRNA effectively reduced IRF-7 mRNA resulting in abrogated type 1 IFN responses with no effect on viral titre (Fig 5A-C). Unlike anti-CCL7, IRF-7 siRNA treatment did not suppress inflammation or chemokine production in the context of an RV induced exacerbation (Fig 5D – H).

FIG 5. Inhibiting IRF-7 suppresses IFN responses during RV induced exacerbation of AAD.

FIG 5

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BALB/c mice were sensitized and challenged to HDM to induce allergic airways disease, then administered with IRF-7 targeting siRNA and subsequently infected with RV to induce exacerbation. Mice were sacrificed 24hrs post infection for analyses. (A) mRNA expression of IRF-7 and (B) positive strand RV1B RNA in blunt dissected airway tissue measured by qPCR. (C) IFN-α and -β in lung homogenates, measured by ELISA. (D) Cell populations present in BALF as assessed by May-Grunwald-Giemsa staining. (E-H) Levels of chemokines and active NF-κB p65 in lung homogenates measured by ELISA. All results are Mean±SEM at 1 dpi (n=3-5 mice per group). *, p < 0.05, **, p < 0.01. ***, p < 0.001. ****, p < 0.0001 as compared to nonsense control group.

Neither CCL7 neutralisation nor IRF-7 inhibition affected lung histopathology

We employed histopathological scoring of 5μm sections of fixed mouse lungs to evaluate pulmonary inflammation in allergic mice based on the presence of perivascular oedema, perivascular and peribronchiolar inflammation, mucus production and alveolar inflammation. All allergic mice displayed high numbers of PAS positive cells as well as perivascular and peribronchiolar inflammatory infiltrates and in some cases, alveolar inflammation. Representative images of RV exacerbation groups are displayed in Fig 6A. RV infection of allergic mice did not increase pulmonary histopathological scores compare to allergic mice given UV control. Furthermore, Inhibiting IRF-7 or neutralising CCL7 during RV exacerbation did not affect pulmonary histopathology scores, although all allergic mice were scored significantly higher than Saline controls (Fig 6B and C).

FIG 6. Inhibiting IRF-7 or neutralising CCL7 does not affect histopathology during RV-induced exacerbation of AAD.

FIG 6

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BALB/c mice were sensitized and challenged to HDM to induce AAD, then administered with IRF-7 targeting siRNA or CCL7 antibodies and subsequently infected with RV to induce exacerbation of AAD. Mice were sacrificed 24hrs post infection for analyses. Lung histopathology showing representative pulmonary inflammation in 5μm slices of fixed lungs with (A) Carbol’s chromotrope-hematoxylin (CR) stains for peribronchiolar inflammation where each image was originally obtained at 200x magnification and is typical of four mice. (B-C) Total histopathological score evaluated using a semiquantitative scoring system described previously (15). All results are Mean±SEM at 1 dpi (n=4-8 mice per group). ****, p < 0.0001 as compared to nonsense or isotype control group.

Discussion

Our study has dissected the role of CCL7 and IRF7 in RV-induced inflammatory and anti-viral responses.

CCL7 activates CC-receptor (CCR) -2, which is expressed on neutrophils to mediate chemotaxis(18). It has previously been shown that CCL7 also binds to CCR3 on eosinophils to induce chemotaxis and activation(19). Airway epithelial cells and macrophages produce CCL7, which may orchestrate cell recruitment in the context of oxidative stress(20, 21). Induction of CCL7 during viral infections has been observed in asthmatic patients(22) and in models of RV infection dissecting the role of CCL2(23). Our study significantly extends these findings by showing that CCL7 inhibition limited macrophage and neutrophil influx into the lung and the development of AHR in non-allergic mice, which was associated with suppression of the MAPK signalling factor ERK1 and NF-κB subunits p50 and p65. In contrast, AHR and NF-κB activation was not significantly reduced by CCL7 inhibition in allergic mice although inflammation was limited. Thus, our data suggests an association between NF-κB activation and AHR, which has been reported previously(10, 11, 24). NF-κB promotes TH2 polarisation and subsequent IL-13 production as well as IgE production from B-cells resulting in mast cell degranulation after allergen-specific IgE cross linkage, both of which directly result in propagation of AHR in the context of allergy(24-28). In the context of RV exacerbations, we have shown previously that by restoring protein phosphatase 2A (PP2A) activity, levels of NF-κB subunits were reduced, which protected against AHR(11). Furthermore, when we inhibited PP2A activity with an siRNA targeted to PP2Acα (the active or catalytic subunit of the polyprotein) in these experiments, both NF-κB activity and AHR were restored(10).

We showed that CCL7-mediated inflammation is not essential for the development of RV-induced AHR in allergic airways disease. Dissociation between AHR and inflammation has been previously reported in other models of allergic airways disease(29) and in subjects with asthma(24, 30).

Interestingly CCL7 neutralisation in RV infection also suppressed IRF-7 and IFN-β expression, which was associated with reduced macrophage but not pDC numbers in the lung. A role of CCL7 in macrophage recruitment is further highlighted by the observation of a correlation between nasal CCL7 production and the recruitment of macrophages in children with virus-induced asthma(31). A previous report showed IFN-α and IFN-β production by macrophages upon norovirus infection, which was abolished in IRF-3 and IRF-7 double knockout cells(32). It is therefore possible that CCL7 increases IRF-7 expression and IFN-β production in the lung by regulation of macrophage inflammation upon RV infection.

To elucidate the role of IRF7 in RV infection we employed siRNA-mediated inhibition. IRF-7 is thought to be a master regulator of type -1 IFN production(33-36) and induction of IRF-7 mRNA has been reported previously(37) in asthmatic children during periods of respiratory viral infections. As expected, IRF7 silencing suppressed IFN-α and IFN-β responses. Interestingly IRF-7 inhibition also reduced neutrophil and macrophage numbers in a similar fashion to CCL7 antagonism but did not reduce AHR or NF-κB activity. While our results therefore support an important role of IRF7 in RV-induced anti-viral responses through induction of IFN production they also reveal an unexpected role of IRF7 in modulating the inflammatory response upon in-vivo RV exposure. Recently IRF7 was identified to control pro- to anti-inflammatory (M1-to-M2) macrophage phenotype switch and IRF7 expression reduced pro-inflammatory macrophage activity(38). This may be relevant to RV infection and the singergistic effects of CCL7 on IRF7 expression observed in our study may regulate balance between the pro-inflammatory activity of lung macrophages and their antiviral capabilities.

Together we dissect the role of CCL7 and IRF7 in RV-induced antiviral and inflammatory responses and show an important collaborative function for the recruitment and activation of inflammatory cells in the lung and for mounting an IFN-β response to RV.

Supplementary Material

1

Acknowledgements

We thank Dr Ana Pereira de Siqueira, Heather MacDonald, Jane Grehan, Leon Sokulsky, Matthew Morton and the staff from Bioresources of the Hunter Medical Research Institute for their technical assistance.

The work in manuscript was funded by NHMRC grant G130093 and funding from Hunter Medical Research Institute (HMRI) and University of Newcastle.

Sebastian L. Johnston and Nathan Bartlett were supported in part by ERC FP7 grant 233015, a Chair from Asthma UK (CH11SJ), MRC Centre grant G1000758 and Predicta FP7 Collaborative Project grant 260895.

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