An inflammatory vascular endothelium-mimicking microfluidic device to enable leukocyte rolling and adhesion for rapid infection diagnosis
Introduction
Extravasation of leukocytes to sites of infection is one of the most critical steps for the successful development of host defense to pathogens. This process is precisely regulated by leukocyte–endothelial cell interactions via several molecules dedicated to activation, recruitment, rolling, and localization of leukocytes (Hickey and Kubes, 2009; Jung et al., 1998). An extravasation cascade of leukocytes is initiated by activation of endothelial cells upon inflammatory stimulation, leading to an upregulation of the molecules participating in leukocyte rolling and adhesion, such as P-, E-selectin, and intracellular adhesion molecule-1 (ICAM-1) (Butcher, 1991; Mayadas et al., 1993; Middleton et al., 2002). It has been reported that leukocytes constitutively express P-selectin glycoprotein ligand-1 (PSGL-1) and E-selectin ligand-1 (ESL-1) (Dore et al., 1993; Martins et al., 2007; Rijcken et al., 2004; Tedder et al., 1995), and are therefore poised to be recruited into an inflammatory lesion instantaneously by engaging P-, E-selectins expressed on activated endothelial cells (Khew-Goodall et al., 1999; Kulidjian et al., 2002). Following the selectin-mediated rolling, lymphocyte functional antigen (LFA)-1 mediates firm adhesion of leukocytes to ICAM-1-expressing endothelial cells for the subsequent extravasation into the infected tissues (Martin and Springer, 1988). Since the leukocyte extravasation process occurs instantaneously when pathogen invasion occurs as a part of the innate immune responses (Kolaczkowska and Kubes, 2013), an ex vivo monitoring of this phenomenon has a great potential on the early diagnosis of sepsis.
Sepsis is a life-threatening illness in which the host immune system causes dysregulated systemic inflammatory response to infection, leading to tissue injury, organ failure, and death (Buras et al., 2005; Rittirsch et al., 2008). Given that the mortality rate associated with sepsis can increase by 8% for every hour of delay in inappropriate antibiotic administration (Kumar et al., 2006), early identification and timely treatment of sepsis are exceedingly critical for improving clinical outcomes (Daniels, 2011; Montaldo et al., 2017; Tissari et al., 2010). However, the traditional diagnosis that mainly depends on a blood culture method (Lambregts et al., 2019) can take up to several days and often results in false positive and false negative outcomes (Benjamin and Wagner, 2007; Hall and Lyman, 2006), even with the supports of state-of-the-art molecular diagnostic tests, such as polymerase chain reaction (PCR) (Marik, 2014), procalcitonin (PCT) (Christ-Crain and Müller, 2005; Farooq and Colón-Franco, 2019) and C-reactive protein (CRP) assays (Nargis et al., 2014; Wu et al., 2017). A diagnostic gap between the “golden hour” of sepsis (Van Zanten, 2014) and a turnaround time of the conventional tests in a clinical laboratory leads to a high mortality rate (30–50%) (Hotchkiss et al., 2017) in hospital and afflicts around 31.5 million people worldwide every year (Fleischmann et al., 2016). These unmet challenges become greatly important when we have to screen a massive number of individuals suspected of the early phase of sepsis because the preliminary screening is often determined by a computerized tomography (CT) scan or PCR (Corman et al., 2020; Huang et al., 2020; Pan et al., 2020), which are neither sensitive enough to detect the early phase of infection nor available for on-site analysis achievable within an hour.
To develop a rapid diagnostic method for sepsis within the critical “golden hour,” we mimicked leukocyte adhesion onto the inflammatory endothelium facilitated by infection and demonstrated the biomimicry in an inflammatory vascular endothelium-mimicking microfluidic system. Leukocyte adhesion has been hardly utilized for developing sepsis diagnostic methods due to the prior knowledge that the leukocyte adhesion is primarily governed by the upregulation of cell adhesion molecules on endothelial cells, not mainly by the associated adhesion ligands present on leukocytes (Harjunpää et al., 2019; Kelly et al., 2007). Surprisingly, we found that PSGL-1 on leukocytes was upregulated upon infection; hence, the increased frequency of leukocytes captured on the CAMs-coated microfluidic channel was predicted to assess the activated immune cells triggered by infection, which differentiate sepsis from a healthy condition as early as an hour after the onset of infection.
Section snippets
Chemicals and reagents
ACK Lysing Buffer (A1049201) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Lipopolysaccharide (LPS) from Escherichia coli O111:B4 purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-Myeloperoxidase antibody [2D4] (FITC) (ab90812) was purchased from Abcam (Cambridge, MA, USA). Alexa Fluor® 488-conjugated PSGL-1/CD162 Polyclonal antibody (anti-PSGL-1 antibody), Alexa Fluor® 555-conjugated Golgi Complex Polyclonal antibody (anti-ESL-1 antibody), Alexa Fluor® 350-conjugated
Characterization of the expression profile of leukocyte adhesion receptors in a rat infection model
We hypothesized that the pro-inflammatory response in infection would upregulate the adhesion receptors on leukocytes that facilitate their rolling and arrest on endothelium underlying an inflamed tissue (Fig. 1a). We predicted that the expression of these receptors would be upregulated by the onset of infection (Fig. 1b), thus promoting more frequent capture of leukocytes on the inflammatory vascular endothelium-mimicking microfluidic channel coated with cell adhesion molecules (Fig. 1c). To
Conclusions
In this work, we developed a microfluidic device that enables rapid determination of infection using a finger-prick volume of blood. We first validated that leukocyte recruitment to the inflammatory endothelium was regulated not only by CAMs overexpression but also by the upregulation of adhesion receptors present on the leukocytes activated by infection. In addition, we demonstrated that infection was accompanied by an increased proportion of leukocytes that express PSGL-1 and ESL-1, which
CRediT authorship contribution statement
Seyong Kwon: Conceptualization, Methodology, Data curation, Writing - original draft. Amanzhol Kurmashev: Data curation, Investigation, Writing - review & editing. Min Seok Lee: Investigation. Joo H. Kang: Conceptualization, Data curation, Writing - review & editing, Supervision.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This research was supported by grants funded by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (Grant No. NRF-2019R1C1C1006124) and the Samsung Research Funding and Incubation Center for Future Technology (SRFC-IT1602-02). S. K. was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF- 2018R1A6A3A11043793).
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These authors contributed equally to this work.