Feasibility and acceptability of point of care HIV testing in community outreach and GUM drop-in services in the North West of England: A programmatic evaluation

Peter Macpherson
Anu Chawla
Kathy Jones
Emer Coffey
Vida Spaine
Ian Harrison
Pauline Jelliman
Penelope Phillips-Howard
Caryl Beynon
Miriam Taegtmeyer
Feasibility and acceptability of point of care HIV testing in community outreach and GUM drop-in services in the North West of England: A programmatic evaluation
R E S E A R C H A R T I C L E Open Access
Background: In Liverpool, injecting drug users (IDUs), men-who-have-sex-with-men (MSM) and UK Africans experience a disproportionate burden of HIV, yet services do not reach out to these groups and late presentations continue. We set out to: increase testing uptake in targeted marginalized groups through a community and genitourinary medicine (GUM)-based point of care testing (POCT) programme; and conduct a process evaluation to examine service provider inputs and document service user perceptions of the programme. Methods: Mixed quantitative, qualitative and process evaluation methods were used. Service providers were trained to use fourth generation rapid antibody/antigen HIV tests. Existing outreach services incorporated POCT into routine practice. Clients completed a semi-structured questionnaire and focus group discussions (FGDs) were held with service providers. Results: Between September 2009 and June 2010, 953 individuals underwent POCT (GUM: 556 [59%]; communitybased sites: 397 [42%]). Participants in the community were more likely to be male (p = 0.028), older (p < 0.001), of UK African origin (p < 0.001) and IDUs (p < 0.001) than participants from the GUM clinic. Seventeen new HIV diagnoses were confirmed (prevalence = 1.8%), 16 of whom were in risk exposure categories (prevalence: 16/517, 3.1%). Questionnaires and FGDs showed that clients and service providers were supportive of POCT, highlighting benefits of reaching out to marginalised communities and incorporating HIV prevention messages. Conclusions: Community and GUM clinic-based POCT for HIV was feasible and acceptable to clients and service providers in a low prevalence setting. It successfully reached target groups, many of whom would not have otherwise tested. We recommend POCT be considered among strategies to increase the uptake of HIV testing among groups who are currently underserved.

Background

The British HIV Association [1] and other guidelines [2,3] recommend that access to HIV testing be expanded and routinely offered in non-specialist settings in order to reach out to high-risk and marginalised sections of the population and reduce late presentations. Point of care testing (POCT) is recommended in settings where venepuncture may not be possible and where there is support from a laboratory confirmatory testing service [1]. Current HIV screening relies on self-presentation to care services, resulting in missed opportunities for early diagnosis, and for preventative counselling of high-risk but currently HIV-negative individuals. United Kingdom Health Protection Agency statistics note that one quarter of the 83,000 HIV-positive individuals in the UK are unaware of their status [4] with at-risk groups particularly vulnerable to late diagnosis [5][6][7]. In Liverpool, three groups share a disproportionate burden of HIV infection: intravenous drug users (IDUs); men who have sex with men (MSM); and people of African origin [4,7].

This study presents findings from a study conducted in Liverpool, United Kingdom, to explore the feasibility and acceptability of POCT for HIV among at-risk and marginalised groups; the success of this approach in reaching out to target groups and previously untested individuals; and to document key factors which impact upon the implementation of POCT for HIV in order to inform others wishing to expand POCT services.

Methods

Mixed methods of research were used incorporating participatory approaches, situational analysis, focus group discussions, analysis of routine patient monitoring data, and analysis of self-completed questionnaires patients recruited for POCT.

Planning and Implementation of POCT for HIV

A multidisciplinary, multiagency steering group was established, comprised of clinicians, specialist HIV community nurses, members of the local primary care trust and public health researchers. Minutes, notes and correspondence following meetings were included in the analysis. Since co-authors were also participants in these groups methods that were cognisant of the observer participant approach were used. One author (PM) was an outsider to the process acting more as observer than participant. Others were active participants whose observations were also considered in the analysis. The steering group undertook situational analysis of a number of existing health and social programmes in Liverpool to identify services and programmes likely to interact with individuals at risk of HIV infection (e.g. outreach care programmes for IVDUs and sex workers) and marginalised people who may find it difficult to access health care (e.g. asylum seekers and homeless people). Services meeting these criteria were invited to participate in the POCT programme, representatives of the management were asked to join the steering group and service providers were trained in POCT for HIV. As the services identified offered a variety of health and social programmes, service providers were encouraged to develop strategies to incorporate POCT into their existing services.

To mobilise interest in POCT, an awareness-raising campaign, called Liverpool Gets Tested, was held around the week of World AIDS Day 2009 (1 st December). This included a number of community-based events such as HIV vigils and radio advertising campaigns.

Service Providers' Experiences of POCT for HIV

Qualitative methods were chosen to elucidate perceptions and experiences as they allow for a more flexible and in-depth exploration of acceptability of new services. The 25 service providers performing HIV POCT at the 6 sites were invited to participate in three focus group discussions (two with providers from community sites and one with providers from the GUM site). All interviews were recorded and transcribed by the first author. A framework approach to analysis was undertaken. Qualitative analysis software (NVIVO8, QSR Software, Melbourne) was used for coding purposes. Emerging themes were grouped and triangulated with the steering committee members. Transcripts were independently reviewed by a second researcher to confirm themes.

POCT Uptake by Service Users and a Survey of Their Perceptions of the Programme

Attendances for POCT at study sites, non-identifiable participant demographic details, and POCT for HIV results (cross-referenced with laboratory confirmatory testing where necessary) were captured by staff in logbooks. Participant demographics and self-reported clinical history were used to code a person as being in a risk exposure category for HIV or otherwise; risk exposure categories included: MSM, IDU, UK African, having bought or sold sex, having been raped, or having an HIV-positive partner. Participants could belong to multiple risk categories. Persons receiving a POCT test for HIV during the week of World AIDS Day were invited to self-complete a questionnaire about their experience of POCT.

HIV-testing Procedures

POCT for HIV was undertaken using Determine ® HIV-1/2 Ag/Ab Combo (Inverness Medical Inc, UK) and tested on fingerprick blood samples. HIV testing and pre-and post-test counselling was conducted in private rooms within each site by nurses who had received training in test kit use and HIV counselling. Rooms had been previously inspected for suitability based on the presence of adequate lighting; a clean, flat and washable testing surface and accessible hand washing facilities. Participants with a reactive or invalid test (that did not resolve on immediate retesting) had a further sample sent for confirmatory same day fourth generation laboratory ELISA testing. The study nurse or clinician conveyed confirmed positive results to the participant in a follow-up appointment and referral for comprehensive HIV care was made to the HIV clinic at the Royal Liverpool Hospital, with the study clinician acting as a link person for accessing care.

Statistical Methods

Client characteristics (logbook) and perceptions of POCT for HIV (Liverpool Gets Tested questionnaire) were expressed as proportions and compared between the LCSH and community-based sites using chi-square tests. The Wilcoxon rank sum test was used to compare differences in age distribution between the groups. Hierarchical risk exposure categories (including mutually exclusive categories with a single, or multiple risk exposures) were constructed for participants testing at the two sites and compared using Fisher's exact test. Statistical analysis used STATA v11.2 (Statacorp, College Station, Tx).

Ethical Approval

Results

Planning and Implementation of POCT for HIV

Through the steering group, six sites were identified as offering health and social care services to target groups. These included a hospital-based GUM clinic (the Liverpool Centre for Sexual Health [LCSH]) and five community-based programmes (a drug-users support group; an asylum-seekers health programme; a MSM health and support programme; a travel clinic; and a support programme for homeless people).

Situational analysis of the study sites showed differences between the hospital-based site (LCSH) and the community-based sites that would influence implementation of the POCT protocol (Table 1). LCSH, an outpatient facility of the Royal Liverpool University Hospital, provides sexual health and HIV services to Liverpool and Merseyside, with over 34,000 attendances in 2009. Clients actively self-present for care and LSCH conducts limited community outreach. POCT was conducted in the department as opportunistic screening for individuals presenting for clinical care.

The five community-based sites, although serving different client groups, had a number of common features relevant in planning the introduction of POCT. In particular, they all had existing social support programmes for the client groups they served. Therefore here it was decided to adopt a more community-orientated approach to POCT. This would involve a focus on awareness-raising about HIV and community mobilisation (including organising church vigils and radio campaigns) to encourage uptake of POCT in target and marginalised groups.

POCT Uptake by Service Users and Their Perceptions of the Programme

Between September 2009 and June 2010, 953 participants underwent POCT for HIV, 556 (58%) at LCSH and 397 (42%) at the community-based sites. During the Liverpool Gets Tested Campaign, 154 people underwent POCT for HIV. Of individuals tested, and where data were recorded, 659/946 (70%) were male and the median age was 29 years (interquartile range (IQR): 23 -38). A greater proportion of people testing in the community-based sites (74%) were males compared to in LCSH (67%, p = 0.028). However, the proportion of males who self-identified as MSM was higher at LCSH (36%) compared to the community-based sites (44%, p = 0.050) Complete risk category data was available for 927 (97%) participants, with all missing data from those testing at community-based sites ( Table 2). In total, 475 (51%) were found to be in one HIV risk category. A further 41 (4%) participants were in two risk categories and 2 (0.2%) were in three risk categories. The most frequently reported category was MSM (28%), followed by UK African origin (14%) and current or previous intravenous drug use (8%). Reflecting the clients groups served by the two models of POCT (community and hospital-based), participants tested in the communitybased sites were more likely to be UK Africans and to report current or previous IDU compared to those tested in LCSH. In contrast, participants tested at the hospital-based LCSH were more likely to have reported having been raped and having been exposed to an HIVpositive partner. Seventeen confirmed HIV diagnoses were made (prevalence = 1.8%); 14 from LCSH (prevalence = 2.5%) and three from community based sites (prevalence = 0.8%). Ten individuals diagnosed HIV-positive were MSM, four were UK Africans, one reported IDU and one reported having been gang raped. One HIV-positive participant had no identified risk category by our definition. HIV prevalence in those in a risk category was 16/517 (3.1%). All but one are under on-going follow up in clinical settings. The remaining one is an IDU with a chaotic lifestyle who has repeatedly failed to attend appointments but remains in the Liverpool area.

During the Liverpool Gets Tested Campaign, 128/154 (83%) of individuals participating in POCT for HIV completed a questionnaire. Risk exposure among those completing questionnaires categories were similar to that of the overall study population, expecting that no participants who reported having been raped or having an HIV-positive partner completed a questionnaire. Approximately half (65/127, 51%) had never tested for HIV before and when questioned about their experience of POCT 105/125 (84%) preferred POCT to laboratory testing and 115/125 (92%) stated they would recommend POCT to others. There were no differences in reported previous testing for HIV (p = 0.867) or preference for POCT between LCSH and the community-based sites (p = 0.759). However, a greater proportion of participants testing at community-based sites (96%) stated that they would recommend POCT compared to those testing at LCSH (82%, p = 0.015).

Process Evaluation of Implementation and Training

Evaluation of minutes of steering group meetings during preparation for and implementation of POCT revealed a number of issues important to others considering multidisciplinary POCT for HIV programmes. The principal concern raised by service providers was that POCT would impact upon their regular duties. This overlapped with concerns from healthcare commissioners that POCT for HIV could distract from core service provision. Furthermore, questions were raised as to how POCT for HIV would continue to be funded following the completion of the research project. The competing demands of the health care commissioners to demonstrate value for money of POCT and that of research academics to demonstrate effectiveness of the HIV testing programme required considerable time and mutual co-operation to resolve. Demonstration that new HIV-positive diagnoses were being made amongst marginalised groups such asylum-seekers and drug users helped persuade commissioners of the value of the POCT.

Service Providers' Experiences of POCT for HIV

Through analysis of focus group discussions, we identified key themes raised by service-providers in LCSH and the community-based sites about their experiences of participating in the POCT for HIV programme.

Community-based service providers

Health care providers working within community-based sites highlighted the benefits of reaching out into the community to increase awareness of POCT for HIV and engaging individuals who may have otherwise not been able to access health services:

"When people come to [the GUM clinic], they already know a certain amount about [HIV] and have thought about it but when we reach out we are tapping out into a whole different community". Nurse, male, drug users outreach programme

The opportunities to integrate POCT with prevention messages for difficult-to-reach clients was noted by service providers who felt that POCT provided a unique opportunity for integrating public health messages:

"They were high risk patients that were non-reactive so it was a good opportunity to reinforce behavioural change ... just reinforce the fact that if he is going to have sex outside of his marriage, particular with high risk, then he needs to protect himself because his whole world will just sort of collapse. So it was worth doing it for that." Community health worker, male, MSM outreach project A number of challenges associated with integrating POCT into community-based service delivery were identified. In particular service providers felt there was a careful line to tread between supporting someone in a marginalised and vulnerable position to learn their HIVstatus and placing undue pressure on an individual who had not planned to take an HIV test.

External quality assurance systems were identified as a key area requiring a strengthened support system. Community-based service providers felt if they obtained reactive or invalid results on POCT, it was difficult to communicate with laboratory personnel and often relied upon informal communications. Delays in obtaining a final HIV result created a period of "diagnostic limbo" for service providers and clients, which negatively impacted upon their relationship.

LCSH-based service providers

Service providers within LCSH highlighted the usefulness of POCT for individuals who were victims of sexual assault and who were about to receive post-exposure prophylaxis. They felt confident offering clients negative results, facilitating rapid reassurance for individuals suffering traumatic experiences. They also acknowledged the benefits of being able to give a rapid result and not having to worry about clients failing to return to the department to collect results.

LCSH integrated POCT into departmental service delivery using a different model to the communitybased sites. Nurses and healthcare assistants (HCAs) undertaking consultations with outpatient clients would offer POCT. If the client agreed, the health care assistants (HCAs)/staff nurses would perform the fingerprick tests themselves. The more complex patients (such as sexual assault cases) were referred to the health advisers. This allowed nurses and HCAs to perform more tests and maximise their busy workload, however, health advisers reported feeling overwhelmed by the difficult cases. We noted that health advisers identified all reactive results in the LCSH and one commented on the impact of dealing with traumatised clients:

"For us it's doing the same procedure again and again and again and everybody that comes has got a different journey to the test. Some of them are incredibly traumatic. Seen a man that was raped yesterday. I've got a prisoner coming in whose been raped. [...] That's the run of the mill for us. It's just too many people." Health advisor, female, LCSH Issues identified by service-providers in both LCSH and community-based sites A common theme for both groups of service providers was their initial concern and confidence in their knowledge of HIV should clients ask difficult questions. These concerns overlapped with the experience of undertaking an activity that was seen as something new and outside their realm of usual practice. Whilst most service providers were experienced in technical procedures and quickly became confident in doing finger-prick tests, many had concerns about how to deal with a positive HIV result and expressed fear at the prospect of giving a result that could have profound and long-lasting implications for the client's health and social life. However, service providers who had given reactive results expressed that they were pleased to enable clients to take control of their illness.

"I think its really good for the nurse or whoever is doing the test to give somebody the result instantly. Because you feel like you are actually [...] helping them achieve something positive, whether it is a negative or a reactive result."

Health worker, male, asylum-seekers health programme Similarly, the giving of negative results had a profound effect on service providers. As confidence and experience grew, service providers recognised the importance of the relationship between the client and themselves and how a negative POCT result represented an opportunity to reinforce educational messages and empower clients to change high-risk behaviour.

Another key theme identified was the speed with which results were obtained. Service providers noted that clients may not have attended hospital or the community site expecting to undertake a HIV test and waiting 20 minutes for the results provided little extra time to receive counseling and understand the implications of the result. Service providers thus found themselves reigning back their enthusiasm for offering clients POCT, in order to ensure clients understood and had time to consider the possible implications:

"Doing the last few tests that I have been doing, I've been saying: look, you are going to get a result quite quickly today you know. Are you prepared for that?" Nurse, female, travel clinic

Discussion

This community-and GUM clinic-based programme has demonstrated that POCT for HIV can reach highrisk, previously marginalised groups, including UK Africans, MSM, homeless people, sex-workers and IDUs, with 953 tests being conducted within a 10 month period, over half of which were among high-risk groups. Of those who had a POCT for HIV during World AIDS Week, over fifty percent had never previously undergone HIV testing.

POCT for HIV has been successfully scaled-up in high prevalence HIV settings (particularly in sub-Saharan Africa), through voluntary counselling and testing (VCT) [8] programmes that provide opportunities to incorporate HIV prevention messages into the testing process [9]. Our findings suggest that POCT in the UK could facilitate discussion of HIV prevention messages, particularly for individuals who are in high-risk categories and who test negative for HIV.

With a third of new UK HIV diagnoses being identified with a CD4 count of less than 200 cells/ul [4], this project demonstrates the potential of incorporating POCT into existing hospital and community based services to increase the uptake of HIV testing, decrease late presentations and increase access for marginalised groups. Late presentation is associated with higher mortality [10,11], prolonged hospital stays and increased hospital-related costs [12].

This study used 4 th generation rapid antibody/antigen tests for HIV. Previous studies in the UK and elsewhere that have introduced POCT to hospital [13][14][15][16][17][18], primary care [13,19] and community settings [20][21][22][23][24][25] have used antibody-only tests. Although 4 th generation tests have the advantage of a shortened window-period and increased sensitivity and specificity [26], we did not identify any seroconverters on the basis of p24 antigen and did have two false positive p24 antigen results. Our findings, which are being presented elsewhere, suggest that 4 th generation test kits do not have an advantage over existing 3 rd generation tests for a community-based screening programme.

We found that service providers and clients were accepting of POCT. The pre-selection of study sites to identify services that would be most successful in providing POCT may have influenced the high observed acceptability for clients and service providers. Previous studies investigating community-based POCT for HIV in the UK raised concerns that lack of confidentiality and stigma surrounding HIV may hinder service uptake [27]. However, we found participants (over 90%) would recommend POCT to others with a greater proportion of participants from community sites than in the GUM setting making the recommendation. There are a number of possible explanations for this. Firstly, the differing client groups (and with potential different spectrum of reasons for presentation) tested may have meant that there was differing levels of what to expect from POCT prior to testing. Alternatively, the way in which testing was offered may have influenced experience of POCT.

In the community, service providers, who may have known the client previously and have a long-standing trusting and supportive relationship with them, offered POCT. In contrast, in the hospital, POCT was likely to be offered by a service provider whom the client was meeting for the first time in the context of a busy GUM clinic appointment.

We also describe positive experiences among service providers, particularly regarding an increase in knowledge and awareness of needs within the community. We attribute this to the decentralised approach taken to allow service providers to integrate POCT into their pre-existing community-programmes.

Establishment of the POCT steering committee and involvement of health-care commissioners in management decisions was critical to the successful scale up. Although we did not assess in this study, it is possible that the availability of POCT may have discouraged some potential testers, Furthers studies to examine the perceptions of individuals who refuse HIV testing towards POCT are required.

In the hospital GUM clinic setting, a large numbers of screening tests were undertaken in a time-pressured environment. As a consequence clients perceived to be "more difficult" were tested by health advisers instead of the HCAs or staff nurses. The emotional impact for service providers of undertaking HIV testing is well recognised [19,23]. We found that the visual emergence of the HIV result to both the client and the service provider to be a difficult experience for service providers. It is important to ensure support and debrief mechanisms exist for staff undertaking rapid HIV testing.

Certain caveats are noted within our study; our participants may represent the most vocal population with particularly strong feelings, either positive or negative, about the service. The high uptake of questionnaire completion (82.5%) during the World AIDS Day study period and our measures to ensure that clients completed questionnaires in private and submitted anonymously attempted to mitigate this. Furthermore, a recognised limitation of focus group discussions is that group dynamics may lead to over-emphasis of certain themes which, in individual interviews, may not have been particularly important. We limited this effect by structuring groups to include service providers working from teams in similar environments and by using flipchart exercises to focus group discussions.

Conclusions

We found POCT to be acceptable to service providers and clients. The decentralised rolling-out of POCT was successfully integrated into a broad range of existing services, and reached diverse groups of clients, including targeted groups. Further cost-effectiveness studies and epidemiological studies are required to assess the additional benefit of 4 th generation POCT over current testing strategies. Together these findings will have implications for further scaling-up of POCT services in Liverpool and more widely in the UK, and for the introduction of additional, novel, point of care tests. Gilead UK and Ireland fellowship programme paid for salary for VS and funded test kits

Funding
PM is supported by the Wellcome Trust Clinical PhD Programme at Liverpool School of Tropical Medicine

Table 1
1POCT Site CharacteristicsLiverpool Centre for
Sexual Health

Community-based sites

Locations
Hospital GUM clinic
Community sites

Model adopted
for POCT

Opportunistic screening
Outreach case finding

Dependent on client
self-presentation to
service

Dependent on service
providers promoting
uptake of POCT

Groups targeted Patients for STI services
Drug users

Sexual assault patients
Asylum-seekers
MSM

Homeless people

Sex-workers

Travellers

UK Africans

Key successes
highlighted by
service
providers

Rapid availability of
results for traumatised
clients

Outreach to encourage
uptake amongst
marginalised and at risk
groups

Universal screening
Giving positive and
negative POCT results to
clients

Integration within nurse
and healthcare assistant
departmental working
patterns

Opportunities for
incorporating prevention
messages

Key challenges
highlighted by
service
providers

Time demands of POCT
Onward client referral
systems

Emotional impact of
performing large
number of HIV tests

Laboratory quality
assurance systems

Need to take further
venous blood samples
(e.g. for syphilis
screening)

Table 2
2Risk categories of participants undergoing POCT and completing "Liverpool Gets Tested Questionnaire" at community-based sites and LCSH * Where data on exposure category captured. § Risk categories are grouped based on hierarchical categories. Any one person with multiple risks may ONLY be represented in the highest category. ± These groups presented are NOT mutually exclusive, meaning a case can be represented in multiple groupings. These summarised categories are meant to give a broader picture of the exposure categories and will NOT add up to the overall total number of participants. † Fisher's exact test.Total (%) Community-based site (%) LCSH (%) P-value †

n = 927
n = 371
n = 556

MacPherson et al. BMC Public Health 2011, 11:419 http://www.biomedcentral.com/1471-2458/11/419 Page 5 of 8
MacPherson et al. BMC Public Health 2011, 11:419 http://www.biomedcentral.com/1471-2458/11/419 Page 6 of 8
AcknowledgementsThe authors would like to acknowledge members of the POCT steering group committee, the Liverpool Blood-Borne Viruses Group and NHS Liverpool. Additional acknowledgement for support go to Robert Downes, Nicholas Beeching, David Lalloo, Peter Carey, Chris Whitehead and Diane Exley. Finally we would like to thank that staff and clients from the community and GUM sites.Authors' contributions PM, AC, KJ, EC and MJ conceived of the study and participated in its design. AC supervised laboratory virological services. VS, IH and PJ undertook data collection. PM and MT undertook data management and statistical analysis. PM, MT, PPH and. CB undertook qualitative data analysis and programmatic evaluation. PM, MT, PPH and CB drafted the paper. All authors read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.
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Implementation of rapid HIV testing programs in community and outreach settings: perspectives from staff at eight community-based organizations in seven U.S. cities. Public Health Rep. H A Clark, K E Bowles, B Song, J D Heffelfinger, 123SupplClark HA, Bowles KE, Song B, Heffelfinger JD: Implementation of rapid HIV testing programs in community and outreach settings: perspectives from staff at eight community-based organizations in seven U.S. cities. Public Health Rep 2008, 123(Suppl 3):86-93.

Expanding HIV rapid testing via point-ofcare paraprofessionals. H Knapp, H D Anaya, J E Feld, Int J STD AIDS. 19Knapp H, Anaya HD, Feld JE: Expanding HIV rapid testing via point-of- care paraprofessionals. Int J STD AIDS 2008, 19:629-32.

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Rapid HIV testing in transgender communities by community-based organizations in three cities. J D Schulden, B Song, A Barros, A Mares-Delgrasso, C W Martin, R Ramirez, L C Smith, D P Wheeler, A M Oster, P S Sullivan, J D Heffelfinger, Public Health Rep. 1233SupplSchulden JD, Song B, Barros A, Mares-DelGrasso A, Martin CW, Ramirez R, Smith LC, Wheeler DP, Oster AM, Sullivan PS, Heffelfinger JD: Rapid HIV testing in transgender communities by community-based organizations in three cities. Public Health Rep 2008, 123(Suppl 3):101-14.

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Pre-publication history The pre-publication history for this paper can be accessed here. 10.1186/1471-2458-11-419Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2458/11/419/prepub doi:10.1186/1471-2458-11-419

Feasibility and acceptability of point of care HIV testing in community outreach and GUM drop-in services in the North West of England: A programmatic evaluation. Macpherson, BMC Public Health. 11419Cite this article asCite this article as: MacPherson et al.: Feasibility and acceptability of point of care HIV testing in community outreach and GUM drop-in services in the North West of England: A programmatic evaluation. BMC Public Health 2011 11:419.


Measurement ofTLR-induced macrophage spreading by automated image analysis: differential role of Myd88 and MAPK in early and late responses
published: 18 October 2011

Jens Wenzel
Immunology and Hygiene
Institute of Clinical Microbiology
University Hospital Erlangen
Friedrich-Alexander Universität Erlangen-Nürnberg
ErlangenGermany

Christian Held
Department of Image Processing and Biomedical Engineering
Fraunhofer Institute for Integrated Circuits IIS
ErlangenGermany

Ralf Palmisano
Department of Biology
Friedrich-Alexander Universität Erlangen-Nürnberg
ErlangenGermany

Stefan Teufel
Medical Clinic 3
Sangdun Choi
University Hospital Erlangen
Friedrich-Alexander Universität Erlangen-Nürnberg
ErlangenGermany

Ajou University
South Korea

Immunology and Hygiene
Institute of Clinical Microbiology
University of Heidelberg
Germany

University Hospital Erlangen
Friedrich-Alexander-Universität Erlangen-Nürnberg, Wasserturmstr. 3-591054ErlangenGermany

Jean-Pierre David
Medical Clinic 3
Sangdun Choi
University Hospital Erlangen
Friedrich-Alexander Universität Erlangen-Nürnberg
ErlangenGermany

Ajou University
South Korea

Immunology and Hygiene
Institute of Clinical Microbiology
University of Heidelberg
Germany

University Hospital Erlangen
Friedrich-Alexander-Universität Erlangen-Nürnberg, Wasserturmstr. 3-591054ErlangenGermany

Thomas Wittenberg
Department of Image Processing and Biomedical Engineering
Fraunhofer Institute for Integrated Circuits IIS
ErlangenGermany

Roland Lang email@example.com
Immunology and Hygiene
Institute of Clinical Microbiology
University Hospital Erlangen
Friedrich-Alexander Universität Erlangen-Nürnberg
ErlangenGermany

Konrad Alexander Bode
Roland Lang
Measurement ofTLR-induced macrophage spreading by automated image analysis: differential role of Myd88 and MAPK in early and late responses
published: 18 October 201110.3389/fphys.2011.00071ORIGINAL RESEARCH ARTICLE Edited by: Kumar Selvarajoo, Keio University, Japan Reviewed by: *Correspondence:macrophagespreadingTLRimage analysis
Sensing of infectious danger by toll-like receptors (TLRs) on macrophages causes not only a reprogramming of the transcriptome but also changes in the cytoskeleton important for cell spreading and motility. Since manual determination of cell contact areas from fluorescence micrographs is very time-consuming and prone to bias, we have developed and tested algorithms for automated measurement of macrophage spreading. The two-step method combines identification of cells by nuclear staining with DAPI and cell surface staining of the integrin CD11b. Automated image analysis correlated very well with manual annotation in resting macrophages and early after stimulation, whereas at later time points the automated cell segmentation algorithm and manual annotation showed slightly larger variation. The method was applied to investigate the impact of genetic or pharmacological inhibition of known TLR signaling components. Deficiency in the adapter protein Myd88 strongly reduced spreading activity at the late time points, but had no impact early after LPS-stimulation. A similar effect was observed upon pharmacological inhibition of MEK1, the kinase activating the mitogen-activated protein kinases (MAPK) ERK1/2, indicating that ERK1/2 mediates Myd88-dependent macrophages spreading. In contrast, macrophages lacking the MAPK p38 were impaired in the initial spreading response but responded normally 8-24 h after stimulation.The dichotomy of p38 and ERK1/2 MAPK effects on early and late macrophage spreading raises the question which of the respective substrate proteins mediate(s) cytoskeletal remodeling and spreading. The automated measurement of cell spreading described here increases the objectivity and greatly reduces the time required for such investigations and is therefore expected to facilitate larger throughput analysis of macrophage spreading, e.g., in siRNA knockdown screens.

INTRODUCTION

Macrophages reside in all tissues and play an important role in tissue remodeling and homeostasis by phagocytosis and digestion of dead cells and cellular debris. Their second function as sentinels for infectious danger is embodied by the expression of pattern recognition receptors for pathogen-associated molecular patterns. The best characterized group of PRR is the toll-like receptor (TLR) family, which includes TLR9 as the receptor for CpG-rich bacterial DNA and TLR4, which together with MD2 forms the receptor for the lipopolysaccharide from the cell wall of Gram-negative bacteria (Kawai and Akira, 2010). Triggering of TLR family members by microbial ligands, e.g., during phagocytosis of bacteria, induces a rapid and massive transcriptional response engendering the inflammatory response to infection. A bottleneck in the signal transduction of TLR is the adapter protein Myd88 that binds to the intracellular TIR domain of most TLR and recruits further adapters (e.g., TRAF6) and kinases (e.g., IRAK1; IRAK4; Kawai et al., 1999;Kawai and Akira, 2010). The major signaling modules activated by TLR are the IKK complex leading to NFκB translocation to the nucleus, and the cascade of mitogen-activated protein kinases (MAPK). MAPK family members expressed in macrophages are ERK1/2, JNK1/2, and p38. These MAPK control the transcriptional response to TLR ligation through phosphorylation-mediated activation of transcription factors (e.g., AP-1, CREB, and many others; Lang et al., 2006); in addition, the plethora of MAPK substrate proteins are involved in diverse cellular processes including cell motility, adhesion, and phagocytosis (Schmidt et al., 2001;Blander and Medzhitov, 2004;West et al., 2004;Kang et al., 2008).

In a recent global and quantitative analysis of TLR4-driven phosphorylation events in primary macrophages, we identified more than 1800 phosphoproteins containing nearly 7000 phosphorylation sites. LPS-stimulation caused reproducible changes in the phosphorylation of around a quarter of all sites both early (15 ) and late (4 h) after stimulation (Weintz et al., 2010). Bioinformatic analysis of the regulated phosphoproteome showed an enrichment of known TLR signaling pathway molecules, but also revealed that genes annotated in Gene Ontology as cytoskeletaland actin-binding proteins were enriched and thus are a hotspot of TLR-induced phosphorylation. The set of cytoskeleton-associated proteins showing phosphorylation after TLR4 triggering includes (among others) adapters like Arp3, Paxillin and Vasp, kinases like Ptk2, and motor proteins like Myo1e, Myo1f, and Myo9b. Some of these proteins possess well characterized functions in phagocytosis, cell spreading and motility in macrophages, or other cell types (Schaller, 2001;Sechi and Wehland, 2004;Kim et al., 2006;Hanley et al., 2010). Others have not yet been associated functionally with cytoskeleton-based macrophage responses.

In order to better understand the control of TLR-driven changes in phagocytosis and macrophage spreading by signaling molecules and components of the cytoskeleton, quantitative readout systems are needed. Ideally, such assay systems should have the potential for high-throughput analysis, for example to test the effects of siRNA knockdown of a larger group of candidate genes. The spreading response of cells to stimulation increases the contact area with the cell culture support material, an effect that can be easily visualized in fluorescence microscopy. Quantitation of spreading responses from many cells can be done manually, e.g., using ImageJ software (Girish and Vijayalakshmi, 2004;Collins, 2007). However, this approach has the drawback of being very labor-intensive and time-consuming, hence excluding the analysis of multiple samples in a reasonable amount of time. In addition, the manual delineation of the cell borders introduces potential bias. In contrast, automatic image analysis has the potential of circumventing these drawbacks and is therefore highly desirable. Available freeware software packages like the so-called CellProfiler (Carpenter et al., 2006) are able to perform an automated image analysis. The software can be adapted to the image content by adjusting the minimum and maximum cell diameter and by choosing a threshold selection method. Furthermore various pre-processing methods exist that can improve image quality. However, the requirement to adjust all these parameters until satisfying results are obtained makes the use very time-consuming for researchers not trained in image processing. Thus, we have developed algorithms to segment nuclei and to determine cell size and contact areas (Held et al., submitted;Held et al., 2011). Here, these methods were applied and optimized to investigate the macrophage spreading in response to TLR4 stimulation and its control by the Myd88 adapter protein and the MAPK family members ERK1/2 and p38.

MATERIALS AND METHODS

MICE

Colonies of C57BL/6, Myd88−/− (Kawai et al., 1999), and p38flox/flox; Mx-Cre (Engel et al., 2005;Bohm et al., 2009) mice were maintained at the Franz Penzoldt Center of the Medical Faculty at the University Erlangen-Nuremberg. p38flox/flox; Mx-Cre mice were injected three times i.p. at week 5 with poly I:C (13 mg/kg body weight) for depletion of p38a in hematopoietic cells as described (Bohm et al., 2009).

REAGENTS

LPS (Escherichia coli 0111:B4) and Cytochalasin D were purchased from Sigma-Aldrich. MEK1 inhibitor was received from Selleck Chemicals. Antibodies to phosphorylated (p)-Erk1/2, p38, p-p38, pMAPKAPK2 (p-MK2) were obtained from Cell Signaling Technology and to Grb2 from BD Biosciences.

MACROPHAGE DIFFERENTIATION

Mouse bone marrow was flushed out of the prepared femurs and tibiae with sterile ice-cold PBS. After 5 min of erythrocyte lysis at room temperature with NH 4 CL [0.15 M] cells were washed in complete Dulbecco's modified Eagle's medium (cDMEM; 0.05 mM β-mercapthoethanol, 1% Pen/Strep; 10% FCS). After overnight depletion of adherent cells non-adherent cells were further incubated in cDMEM containing 10% L-cell conditioned medium (LCCM) as a source of M-CSF for 6 days on 10 cm bacteriological plates at 37˚C, 5% CO 2 . After 3 days in between 5 ml of cDMEM + 10% LCCM was added to the cell culture.

STIMULATION OF MACROPHAGES

Differentiated macrophages were washed with 10 ml sterile PBS, incubated with accutase, collected and washed twice with cDMEM. Macrophages were seeded at a density of 5 × 10 4 and 2.5 × 10 4 cells/well on eight-well Permanox chamber slides (Nalge Nunc International). After an overnight cultivation at 37˚C cells were stimulated with 100 ng/ml LPS for different time points and finally fixed for 20 min with 2% PFA. Supernatants of stimulated cells were collected for examination of cytokine secretion by ELISA.

WESTERN BLOT

To determine the efficiency of MEK1 inhibitor and p38 deletion, parallel cultures with 1 × 10 6 cells/well were cultured in 1 ml cDMEM in 12-well plates. Cell lysates were prepared at the indicated time points after LPS-stimulation. 25 μl of lysate were loaded on 10% PAA gel containing SDS. After blotting membrane was blocked in TBS buffer containing 3% BSA and 0.01% Tween20 and proteins were detected with the respective antibodies.

ELISA

Supernatants from the spreading experiments were collected from the respective conditions and cytokine concentration was measured by ELISA kits from R&D. Samples were treated as described in the manufacturer's instructions.

STAINING PROCEDURE

For visualization of the cell surface by fluorescence microscopy, macrophages were incubated for 30-45 min at room temperature with 1 μg/ml Allophycocyanin (APC)-labeled anti-mouse CD11b antibody (BioLegends) in PBS containing 2% FCS. Cells were washed twice with PBS and nuclei were stained by addition of 1 μg/ml DAPI (Sigma D8417) in PBS and incubation of 10 min at room temperature. For further sample preparation cells were washed again with PBS, mounted with 70% glycerol on specimen and covered.

Frontiers in Physiology | Systems Physiology

IMAGE ACQUISITION AND PROCESSING

Images were acquired using a Zeiss AxioVert 200M (Germany) widefield microscope connected to an AxioCam MRm camera. Images were taken using a 20× objective and post-processed by contrast and brightness enhancement within the AxioVison 4.8.2 software (Carl Zeiss MicroImaging). CD11b-APC stained macrophages were imaged using a CY5 filter set from AHF (Germany).

ALGORITHM DESCRIPTION

Staining of the cell nuclei facilitates the unequivocal definition of individual cells in macrophage cultures containing cells in contact with each other. This information about the nuclei was also included for automatic segmentation, i.e., the definition of macrophage cell bodies against the background and from other cells. The resulting two-step segmentation method consists of the segmentation of all cell nuclei based on the DAPI stain, followed by the segmentation of the macrophages according to the CD11b cell surface signal.

SEGMENTATION AND SEPARATION OF NUCLEI

For the automatic segmentation of the nuclei, a watershed transform-based segmentation routine (Roerdink and Meijster, 2000) was applied. For pre-processing, Gaussian smoothing was applied to reduce the noise level in the image. After shading correction, an additional pre-processing filter is applied which facilitates the splitting of touching cell nuclei. This algorithm will be described in detail elsewhere (Held et al., submitted). In brief, this filter uses adaptive weighting of the local principal curvature C P . As boundaries between touching cells usually hold C P > 0, intensities of these pixels are reduced while other pixels are preserved. The result of this operation is denoted as modified curvature C − P :
C − p = C p, if C p > 0 0, else .
After normalization to the range of [0,1] the modified curvature C − P can be used as a weight map for the input image I, yielding a filtered image I F :
I F = α + (1 − C − P ) p 1 + α ,
where the parameters α and p determine the strength of the applied filter. After filtering, the nuclei are separated from the image background by a k-means clustering-based threshold selection method (Hartigan and Wong, 1979). Holes in the resulting binary image were filled and a distance transform (Saito and Toriwaki, 1994) was applied to incorporate prior knowledge on the morphology of the nuclei. For separation of nuclei touching each other a watershed transform was applied to the distance image.

SEGMENTATION OF CELLS

After segmentation of the cell nuclei, the contact area of the macrophage with the slide was segmented. Details on this algorithm have been described elsewhere (Held et al., 2011). In brief, analog to the nuclei image, the macrophage image was smoothed with a Gaussian filter and a shading correction was performed. Afterward, the cells were separated from the image background by application of the k-means clustering algorithm. Note that a different number of clusters was used for the separation of "background and cells" and "background and nuclei." For separation of the cells a gradient magnitude based fast marching level set method (Sethian, 1999) was performed, using the segmented cell nuclei as initialization.

STATISTICAL ANALYSIS

If not described otherwise in the figure legend, results were expressed as means ± SEM of at least 300 cells per condition. Graphs were generated with GraphPad Prism and statistical significance was determined with Student t test for unpaired conditions (ns = not significant; * p < 0.05; * * * p < 0.0001).

RESULTS

To quantitatively measure spreading responses of macrophages to TLR stimulation, the contact area of the cell to the support material needs to be determined. In order to define the cell borders against the background of the slide, we tested different staining approaches for the best discrimination. In our hands, using bone marrow derived mouse macrophages cultured on Permanox chamber slides, the staining of the integrin CD11b with an APC-labeled antibody resulted in more even staining than the lipid-staining molecule PKH and in a better signal-to-noise ratio than the cytosolic dye CFSE (data not shown). Therefore, CD11b staining of the macrophage cell surface was used for definition of the contact area to the slide and combined with a DAPI staining of the cell nuclei to clearly identify individual cells on fluorescent microscopy images (Figure 1A,B). The spreading response of macrophages to stimulation with the TLR4 agonist LPS was examined in a kinetic analysis using time points between 1 h and 24 h ( Figure 1C). A small but significant increase in the contact area of 10-15% was observed already 1 h after addition of 100 ng/ml LPS. Over time, macrophages continued to spread on the Permanox surface and extended the contact area to approximately twice the initial size after 8-24 h. In most experiments, the maximum effect was observed at 24 h; in some experiments, a peak was reached already 8 h after LPS-stimulation (see below). The increase in the macrophage contact area with the Permanox surface was completely prevented when the inhibitor of actin polymerization Cytochalasin D was added to the cultures before addition of LPS ( Figure 1C). The manual annotation of contact areas from the fluorescence microscopy images is very time-consuming, prohibiting the performance of experiments with multiple conditions. Therefore, we applied the automated two-step segmentation algorithm described in the Section "Materials and Methods" to the raw image data from resting and LPS-stimulated macrophages (Figure 2). The results of the automated segmentation are displayed by the software and the annotation of the cell borders is highlighted (Figure 2B), allowing quality control and manual editing by the user (Figure 2C). While for most cells, the segmentation obtained by the software was found to be correct upon inspection, in the case of overlapping cells some manual editing of the contact area annotation was required (arrows in Figure 2C). www.frontiersin.org To test the accuracy and reliability of the automatic segmentation algorithm, we directly compared the distribution of contact areas on a large number of macrophages under resting and LPS-activated conditions using automated segmentation by the software tool versus manual annotation as the gold standard ( Figure 3A). In this comparison based on the image data from a single experiment, both methods yielded very similar distribution of contact areas and median values in resting cells and early after LPS-stimulation. At the later timepoints, the automatic segmentation algorithm produced a "shrinkage effect" with a smaller median value compared to the manual annotation. To determine whether this "shrinkage effect" is generic at later timepoints, we extended this comparison analysis to a series of six independent experiments ( Figure 3B). Comparison of the median contact area values shows a consistent high agreement between the automated method and the manual annotation in resting macrophages and early (2 h) after stimulation. At the later timepoints, the differences obtained by both methods tended to become larger; however, in addition to "shrinkage effects" there were also "blow-up effects," i.e., examples where automatic annotation gives larger values compared to the manual annotation method. Overall, we observed a good agreement between the results obtained by automated segmentation and manual annotation even at the later timepoints. Therefore, we employed the automated method to investigate TLRtriggered macrophage spreading and its control by canonical TLR pathway molecules; for comparison and validation of the method, the time-consuming manual editing of the automatic annotated data was included in each experiment.

Most TLR employ the adapter protein Myd88 for activation of the major signaling pathways leading to gene expression and cytokine secretion. The MAPK p38 and ERK1/2 are activated by TLR stimuli and contribute to cellular responses through phosphorylation of transcription factors and other substrate proteins. We employed macrophages with genetic deletion of Myd88 and p38, and pharmacological inhibition of the ERK1/2 kinase MEK1, to determine the control of LPS-induced macrophage spreading by these molecules. The effect of genetic and pharmacologic deletion on the cytokine response to LPS was examined using supernatants from the cultures used for analysis of cell spreading (Figure 4). In the absence of Myd88, secretion of the pro-inflammatory TNF, IL-6, and IL-12p40, as well as of anti-inflammatory IL-10 was almost completely absent. In contrast, the inhibition or genetic Frontiers in Physiology | Systems Physiology FIGURE 2 | Segmentation and annotation of macrophages with two-step segmentation software. Fluorescence microscopy images of CD11b-APC-and DAPI-stained macrophages were uploaded into the software (A), nuclear segmentation and contact area annotation was performed by the software tool (B), and finally checked and corrected by the user (C). Arrowheads indicate shrinkage effects; arrows point to overlapping cells necessitating manual editing of the annotation. Cells were imaged by a 20× objective. Scale bar represents 50 μm. ablation of the MAPK family members had more subtle effects. TNF is secreted rapidly after LPS-stimulation; its levels were moderately (40-60%) decreased by the MEK1 inhibitor PD184352 or in p38-deficient macrophages throughout the time course ( Figure 4A). In contrast, IL-6 is secreted later and was less affected by interference with MAPK signals (Figure 4B). Of interest, IL-12p40 was increased upon inhibition of MEK1 or in p38-deficient macrophages ( Figure 4C). IL-10 production was severely impaired in the absence of p38, but also blocked considerably by MEK1 inhibition (Figure 4D).

We next determined the spreading response of macrophages after LPS-stimulation in the absence of Myd88 (Figure 5). In a comparison of Myd88 heterozygous and knockout macrophages, we observed that the strong induction of macrophage spreading at the late 8 and 24 h timepoints was indeed severely impaired in the absence of Myd88. However, Myd88−/− macrophages did respond nearly as well to LPS at the early timepoint of 2 h as the heterozygous control cells. Although there were slight differences in the absolute values, this pattern of responsiveness was robustly identified by both segmentation methods (left and right panel in Figure 5). Myd88−/− macrophages showed a complete lack of cytokine secretion when supernatants from the chamber slide cultures were examined by ELISA (Figure 4). The conserved early spreading response in the absence of Myd88 is therefore likely independent of cytokine secretion. Furthermore, it indicates that other signaling molecules (e.g., the adapter TRIF) may play an important role in cytoskeletal rearrangement after LPS.

The second generation MEK1 inhibitor PD184352 has been reported to have a higher specificity than the older reagents U0126 and PD98059 for MEK1 over MEK5 (Grill et al., 2004), and was therefore used here. Pretreatment of macrophages with PD184352 strongly reduced the levels of basal and LPS-induced phosphorylation of ERK1/2 without obvious impact on the phosphorylation of p38 MAPK ( Figure 6A). MEK1 inhibition had no effect on basal macrophage spreading, and did not change the significant spreading observed at the 2 h timepoint (and in the single experiment also at the 1 h and 4 h timepoints). However, the further increase in spreading at the 8 h and 24 h timepoints was prevented; in fact, similar to the effect of Myd88 deficiency, MEK1 inhibition caused a reduction of the macrophage contact area between 8 h and 24 h ( Figure 6B). Again automatic annotation and manual editing gave basically identical results.

A possible contribution of the p38 MAPK to LPS-induced macrophage spreading was investigated using macrophages derived from the bone marrow of conditional p38flox/flox; Mx-Cre mice treated with poly I:C to induce deletion of p38. As shown in Figure 7A, deletion of p38 was very efficient in mice expressing Cre; consequently, phospho-p38 was undetectable. In contrast, the early activation of ERK1/2 was unchanged in the absence of p38, whereas at later timepoints an even stronger ERK1/2 activation was apparent. The spreading of macrophages in response to LPS was only moderately affected by the absence of p38 (Figure 7B). At the early 2 h timepoint, p38−/− macrophages showed a significantly attenuated increase in the contact area. This effect was seen with the automatic annotation method as well as after manual editing of the segmentation results. However, by 8 h and 24 this difference between p38−/− and p38+/+ macrophages had nearly vanished www.frontiersin.org and p38−/− macrophages displayed similar spreading behavior as p38+/+ cells. The 8 h time point in this experiment is the only instance, where the slight variation between the automated image analysis and the manual editing method led to a different result, in that a significant effect of p38-deficiency was found with the automated annotation but not after manual editing. Together, p38 appears to be required for maximal early spreading, but in marked contrast to the strong effects observed for Myd88 deficiency and inhibition of ERK1/2 activation, p38 is not involved at later timepoints.

DISCUSSION

In this manuscript, we have described the application of a newly developed algorithm for automated image analysis in the investigation of macrophage spreading in response to TLR stimulation. The rapid acquisition of quantitative and reliable data from microscopy-based assays of changes in cell size and adherence is necessary for the comprehensive investigation of the typically relatively large number of candidate genes identified in systems approaches like transcriptome or proteomic screens. We report here that the algorithm developed, a combination of nuclei separation filter based on a watershed segmentation with subsequent cell segmentation by fast marching level set (Held et al., 2011), performed very well in resting macrophages and early after stimulation; at later timepoints an increase in variation was observed. However, our comparison of the automated algorithm with manually corrected annotations showed that overall very similar results were obtained across several experiments investigating the effects of perturbations in TLR signaling on macrophage spreading. Thus, at least for the relatively strong effects on macrophage spreading observed here, the two-step segmentation method presented and tested here can be used without the need for manual editing of the data, leading to a tremendous reduction in the time and labor required to obtain quantitative data on macrophage spreading.

Hence, using this method medium and high-throughput analysis of macrophage spreading appear feasible. For the investigation of more subtle differences, and for validation of effects found with the automated analysis tool, manual inspection, and editing of the annotation may be required. Of note, even with such a semi-automated method of combining tool-based annotation with manual editing the processing time of the microscopy data is reduced by a factor of two to three compared to manual annotation of cells, thereby enabling the investigation of much larger data sets in a reasonable time frame. Taken together, we are convinced that the method described here represents a considerable technical advance and valuable addition to the toolbox required for quantitative, unbiased, and automatic image analysis of innate immune cells.

To further improve the performance of the automatic segmentation tools several issues should be addressed in future work. First, the quality of the input data, i.e., fluorescence microscopy pictures, in terms of intensity of staining and signal-to-noise ratio of cells versus slide background, appears to be the most critical parameter. We have compared the cytosolic dye CFSE, the membrane lipid stain PKH and APC-labeled anti-CD11b staining (data not shown) and obtained the best results with surface molecule staining by anti-CD11b antibody. The reduced accuracy of the annotation tool at later time points after stimulation may be related to changes in CD11b surface expression and/or redistribution of CD11b in different cellular compartments, creating internal maxima that are falsely recognized as cell borders (Figure 2B,C). Thus, staining of other surface markers with strong expression independent of the activation status could lead to enhanced performance of the tool; possible surface markers on macrophages could be MHC-I, CD45, or Fc receptors. In addition, the use of fluorescent dyes yielding stronger signals for labeling of antibodies will be tried to increase signal-to-noise ratios. Another difficulty is the automatic segmentation and annotation of overlapping cells, which may be solved by further development of the segmentation algorithm.

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TLR4-triggered macrophage spreading on Permanox slides induced by stimulation with LPS was observed first after 1 h and increased steadily until 8-24 h. The spreading response could be a consequence of direct TLR-driven signals causing actin polymerization and cytoskeletal rearrangement, or it could depend on indirect effects of TLR-induced secretion of cytokines, e.g., TNF or IL-1. We have not dissected these possibilities here; to do so will require the use of inhibitors of protein synthesis (e.g., Cycloheximide), transport and secretion (e.g., Brefeldin A), or specific interference with certain cytokines by using knockout macrophages. By comparing the kinetics of secretion of the cytokines TNF, IL-6, and IL-12p40 with the spreading response, we observe that only TNF is produced early enough to have a potential role in the initial spreading response. To assess the role of TNF in macrophage spreading, inhibitors of the metalloprotease Adam17 (also known as TACE) could be used.

The central adapter protein of TLR signaling, Myd88, is required for LPS-induced cytokine production (Figure 4) and for the late and enhanced increase in the contact area; however, the early spreading response at 2 h was surprisingly normal in Myd88−/− macrophages (Figure 5). To our knowledge, macrophage spreading of Myd88−/− macrophages has not been analyzed before. Since TLR4 utilizes in addition to Myd88 the adapter protein TRIF (Yamamoto et al., 2003;Weighardt et al., www.frontiersin.org

2004)

, signaling via this pathway may be responsible for the early increase in contact area. Macrophages from mice deficient in Trif or in both Myd88 and Trif will be useful to formally test this possibility. In addition, to differentiate between direct effects of Myd88 on macrophage spreading and indirect effects via secreted cytokines, transfer of supernatants from WT macrophages onto Myd88−/− macrophages should be informative. The MAPK p38 and ERK1/2 are activated by TLR4 signaling and were therefore investigated for a role in LPS-induced macrophage spreading. An unexpected dichotomy in the requirement for p38 and ERK1/2 for early and late spreading responses, respectively, was found (Figures 6 and 7). The spreading response in cells treated with the MEK1 inhibitor PD184352 was very similar to that of Myd88−/− macrophages, with intact spreading at 2 h but a strongly reduced response after 8 and 24 h. Hence, it appears that ERK1/2 is the main kinase of the Myd88-dependent spreading response. A caveat to consider here is the limitation of pharmacological inhibitors: first, PD184352 did not completely suppress the initial ERK1/2 phosphorylation (although strongly diminishing basal and induced expression), and secondly, it may have additional effects independent of MEK1 inhibition that could contribute for the observed late inhibition of spreading. Therefore, a definitive experiment to test the role of ERK1/2 will require the use of macrophages deficient in these MAPK proteins. This genetic approach was used here for p38 MAPK, with apparently complete deletion of p38 after poly I:C injection. In the absence of p38, a reduced early, but intact late spreading response was found. It is worth mentioning that p38-deficient macrophages, similar to Myd88−/− macrophages, had a slightly reduced basal contact area (Figures 5  and 7), which could also indicate differences in macrophage differentiation in the absence of Myd88 and p38. To corroborate the p38-dependence of early LPS-induced spreading, specific pharmacological inhibition of p38 in future experiments will be helpful. Such experiments have already been performed in a macrophage cell line, showing that p38 induces integrin-dependent spreading of J774 macrophages via activation of the Ras-like GTPase Rap1 (Schmidt et al., 2001). As this early defect was not seen in Myd88-deficient macrophages, the logical implication of the data is that p38 activation should be independent of Myd88. In fact, the LPS-induced phosphorylation of p38 and ERK1/2 MAPK is only somewhat delayed in Myd88-deficient macrophages (Kawai et al., 1999).

Which adapters, GTPases and motor proteins are involved in the LPS-induced spreading response, and how are they controlled by the Myd88-and MAPK-dependent pathways described here? One established pathway from TLR4 to integrin activation is via activation of the focal adhesion kinase-related Pyk2 and the adapter protein paxillin (Williams and Ridley, 2000). Paxillin activation and cell spreading in response to LPS involves ERK1/2dependent phosphorylation of Ser130 which is a prerequisite for www.frontiersin.org GSK3-dependent phosphorylation of Ser126 (Cai et al., 2006). All members of the ERK1/2-GSK3-Pyk2-Paxillin module were found strongly phosphorylated after LPS at multiple sites in our published phosphoproteome analysis (Weintz et al., 2010). Of interest, paxillin and Pyk2 activation were only partially reduced in Myd88deficient macrophages (Hazeki et al., 2003), consistent with their potential role in the intact initial spreading in Myd88-deficient macrophages observed here. The observation that Cdc42 and Rac GTPase activation proceeds independent of Myd88 early after LPS-stimulation (Kong and Ge, 2008) provides another potential mechanism for macrophage spreading. Clearly, while some of the players in macrophage spreading are well established, the regulated phosphorylation of more than 40 proteins with a Gene Ontology annotation of "actin-binding" or "cytoskeleton-binding" (Weintz et al., 2010) indicates that multiple proteins contribute to the changes in macrophage shape, contact area and motility after TLR stimulation. Elucidation of the functional role of these proteins will require siRNA knockdown experiments, macrophages from knockout mice and the use specific pharmacological inhibitors. We believe that the method of semi-automatic measurement of macrophage spreading will greatly facilitate the timely, unbiased and quantitative investigation of these perturbations.

FIGURE 1 |
1LPS-induced spreading of macrophages. BMM were plated in eight-well chamber slides and rested overnight before addition of LPS (100 ng/ml). The inhibitor of actin polymerization Cytochalasin D (5 μg/ml) was added 1 h prior to LPS-stimulation. At the indicated time points after addition of LPS, slides were processed for staining of CD11b and nuclei, followed by fluorescence microscopy. (A,B) Representative images from control and 24 h time points. Scale bar = 50 μm. (C) Quantitation of cell spreading by manual annotation of CD11b staining. Shown are mean and SEM of at least 300 cells per condition from one representative experiment. Media control (open circles), LPS (closed circles), and LPS in the presence of Cytochalasin D (closed squares). Statistical significance refers to LPS-treated compared to untreated condition. ns = not significant; * p < 0.05; *** p < 0.0001.

FIGURE 3 |
3Quantitative comparison of manual versus automatic analysis versus automatic with manual editing. (A) Comparison of cell contact area measured automatically versus manual annotation. Data are from one experiment. Each dot represents one cell. Black bars within the scatter plot show median contact area (numerical value indicated above). (B) Comparison of data obtained by annotation with tool (open circle) versus combination of tool and manual correction (closed circle) from six independent experiments and four experimental conditions. Each dot represents the median of at least 300 annotated cells.

FIGURE 4 |
4Control of LPS-induced cytokine secretion by Myd88, ERK1/2, and p38. Supernatants of the cultures used for macrophage spreading analysis were analyzed by ELISA for the presence of the cytokines TNF (A), IL-6 (B), IL-12p40 (C), and IL-10 (D). To normalize for differences between experiments in the absolute amount of cytokines produced, the maximum level of the LPS-treated WT or control macrophage was set to 100% (shown as --). Shown are mean + SD values from two independent experiments.

FIGURE 5 |
5Phenotype of Myd88−/− macrophages in cell spreading. BMM from Myd88± and Myd88−/− mice were plated and stimulated as described in Figure 3. Macrophage spreading was analyzed by automated image analysis (left panel) and manual editing (right panel). Shown are mean and SEM from one representative experiment of two performed. LPS (closed symbols), media control (open symbol), Myd88± (circles), Myd88−/− (squares). Statistical significance refers to Myd88± compared to Myd88−/− genotypes. ns = not significant; *** p < 0.0001.

FIGURE 6 |
6Role of the MAPK ERK in macrophage spreading. BMM from C57BL/6 mice were plated as described in Figure 3. (A) Western blot control for ERK1/2 and p38 phosphorylation. Cell lysates were taken at indicated time points (15 min to 24 h) from two individual experiments. Grb2: loading control. (B) Effect of pharmacological blockade of ERK1/2 activation on spreading. Macrophages were pre-incubated with the MEK1 inhibitor PD184352 (10 μM, squares) or not (circles) for 1 h prior to addition of LPS (closed symbols) or media control (open symbols).Contact area data obtained by automated image analysis (left panel) and manual editing (right panel). Statistical significance refers to PD184352-treated compared to no inhibitor. ns = not significant; *** p < 0.0001.

FIGURE 7 |
7Role of the MAPK p38 in macrophage spreading. Bone marrow from p38flox; Mx-cre; and p38flox mice treated with poly I:C to induce deletion of the p38 gene was used to generate BMM. These cells were plated and stimulated as described inFigure 3. (A) Western blot control for p38 protein levels and phosphorylation of p38 and ERK1/2. Cell lysates were taken at indicated time points (15 min to 4 h). Grb2: loading control. (B) Macrophage spreading kinetics of Cre+ (KO, squares) and Cre− (WT, circles) BMM stimulated with LPS (closed symbols) or media control (open symbols). Results of automated image analysis (left panel) and manual editing (right panel). Statistical significance refers to the comparison of p38-deficient versus WT macrophages. ns = not significant; *** p < 0.0001.
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ACKNOWLEDGMENTSThis project received funding from the Deutsche Forschungsgemeinschaft (SFB 643, TP A10; and SFB 796, B6, A4, and Z). The authors thank Katrin Jozefowski for mouse genotyping, and Matthias Engelbrecht for advice with statistics.Conflict of Interest Statement:The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Received
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