Inflammation

Overview:

Inflammation is a fundamental immune process in that it serves as the body’s natural response to infection and injury. Take, for instance, a cut to the skin. Compromising this barrier to the outside world almost inevitably introduces pathogens, such as bacteria, into tissues. The inflammatory arm of the immune system responds by releasing pro-inflammatory cytokines, which act on local blood vessels to recruit other immune cells to the area, resulting in swelling and redness. Inflammation also aids in limiting the spread of infection and initiates recovery and wound healing.

There are a number of inflammatory biomarkers. Here, the focus will be on key proinflammatory proteins that are often measured in human studies. Cytokines including tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, and (IL)-6 are released from immune cells (e.g., T cells, activated macrophages), fat, and muscle, among other cells and tissues. The acute phase protein C-reactive protein (CRP) is also an inflammatory biomarker released by the liver. The temporal sequence of inflammatory signaling by these biomarkers is in this order: TNF-alpha, IL-1, IL-6, and finally CRP.

Acute inflammation, as described above, serves a critical function. In contrast, elevated concentrations of proinflammatory proteins in blood have been linked to chronic health conditions including cardiovascular disease, cancer, type 2 diabetes, and depression, as well as frailty, loss of bone and muscle mass, and neurodegenerative diseases in older adults. Given the associations between stress and chronic health conditions, inflammation has been posited as a key biological pathway through which psychological factors, including stress, may contribute to disease risk.

Links between stress and inflammation: There is fairly consistent evidence that stress, both acute and chronic, is related to elevations in inflammatory biomarkers. These findings are particularly robust for studies of acute laboratory stress, which have demonstrated a significant stress-related increase in the blood concentrations of IL-6, TNF-alpha, and IL-1beta (another key proinflammatory cytokine), but not CRP (Marsland et al., 2017; Steptoe et al., 2007). Chronic stress has also been linked to higher levels of inflammatory markers in the blood (Miller et al., 2009; Segerstrom and Miller, 2004), though here the findings are somewhat more mixed and may be dependent on the type and severity of the stressor as well as characteristics of the population. 

 

Collection and Measurement

Circulating levels in blood: Circulating levels of proinflammatory cytokines and CRP are typically measured in blood and, if possible, a fasting blood draw is recommended. Once the blood is drawn, it should be kept on ice, processed, and the serum/plasma frozen at -80C until assay. There are circadian rhythms to some proinflammatory cytokines that are the mirror image of those in cortisol, albeit less pronounced (Meier-Ewert et al., 2001). Therefore, samples of blood or saliva should be taken at the same time of day if possible and the time of day recorded and statistically adjusted if not.

 

Dried blood spots: Dried blood spots offer an alternative way of assessing systemic levels of inflammatory mediators and are useful for situations when it is difficult to conduct venipuncture to obtain blood samples (e.g., when studying children or collecting samples in the field). Dried blood spots are obtained quickly and easily by pricking the tip of the finger with a lancet, and allowing blood to drip from the cut onto a collection card; passive dripping is preferred, as squeezing the finger can induce a local inflammatory response. Once obtained, the collection cards are allowed to dry and stored at -20C until assayed. Notably, with some training, dried blood spots can be collected by study participants, making them useful for epidemiologic studies and for repeated momentary assessments in real time. Evidence shows high correlations between serum and dried blood spot levels of CRP (r = 0.96; McDade, Burhop, & Dohnal, 2004; Villalba et al., 2019), validating the use of dried blood spots as a method of assessing systemic levels of inflammation. Preliminary results suggest that circulating levels of IL-6 can also be reliably measured using dried blood spots (Miller & McDade, 2012).

Saliva: Because blood is often hard to obtain in certain populations, e.g., children, investigators have turned to measuring proinflammatory cytokines in saliva. Although many inflammatory proteins can be reliably measured in saliva, the correlations between circulating levels of proinflammatory mediators in blood and saliva have generally been poor (Slavish et al., 2015). It is likely that levels of inflammatory markers in saliva reflect the oral environment as well as systemic inflammation (Fernandez-Botran et al., 2011; Out et al., 2012). Additionally, many inflammatory proteins may be too large to easily infiltrate from the blood into saliva through passive diffusion (Bosch, 2014). Salivary proinflammatory cytokines are also more variable than serum proinflammatory cytokines, either because measurement reliability is not as high or because the oral environment changes more than the systemic environment (Segerstrom, 2020).

Assays for blood, dried blood spots, and saliva: Whether assessed in blood, saliva or using dried blood spots, proinflammatory proteins can be quantified using enzyme-linked immunosorbent assay (ELISA), multiplex arrays (e.g., Luminex, Meso Scale Discovery), or flow cytometry (when looking at inflammatory proteins inside of cells). There is growing interest in the use of multiplex assays, which quantify levels of multiple inflammatory markers from a single small amount of sample. Although cost effective, these assays are not always as sensitive as other methods and the reliability of these systems is highly variable, with some multiplex analytes correlating better with ELISA results than others. In healthy populations with low systemic concentrations of inflammatory biomarkers, high sensitivity methods, such as ELISA or standardized immunoassay (e.g., those offered through the Ella platform from ProteinSimple) are required to reliably detect levels. 

Strengths and weaknesses: Measures of circulating levels of proinflammatory cytokines are commonly associated with sociodemographic, behavioral, and psychological measures of interest to social and clinical scientists. They are fairly easily obtained and, when paired with other risk factors, potentially provide a meaningful index of biological risk. In healthy individuals, changes in these measures may signal response to environmental demands, whereas in medically compromised participants, they may indicate change in disease status. Evidence that inflammation is the causal mechanism in disease is clearer for some diseases than others; for example, there is more support for inflammation as a part of a causal pathway for cancer, cardiovascular disease, and depression. For diseases that have a less established causal pathway, inflammatory markers are often viewed as more of a biomarker than a causal mediator. A primary limitation of interpreting circulating levels of proinflammatory mediators is lack of clarity regarding their biological source (immune cells, fat, muscle), although some biomarkers (e.g., IL-6) have more sources than others (TNF-alpha). Thus, it is possible that increases in proinflammatory cytokines (e.g., IL-6) may be driven by SNS or HPA regulation of immune cells (Irwin and Cole, 2011), or by adipocytes, which have different implications for understanding links between stress and inflammation. Another limitation is that proinflammatory mediators, with the exception of CRP, show marked diurnal variation (Meier-Ewert et al., 2001). As such, researchers interested in measuring systemic levels of proinflammatory cytokines must account for this variation, often by collecting these blood measures within a predetermined window (e.g., between 9-11AM).

 

Additional Measurement Methods

Stimulated cytokine production is an alternative way to measure levels of proinflammatory activity. Unlike circulating proinflammatory biomarkers, this provides an estimate of the functional capacity for cells to produce proinflammatory cytokines when stimulated by a pathogen. Like circulating proinflammatory biomarkers, stimulated cytokine production is also sensitive to acute laboratory stress (Marsland et al., 2017). The procedures for carrying out this assay vary from laboratory to laboratory but for initiating proinflammatory cytokine production, investigators typically incubate the sample with lipopolysaccharide (LPS) for a period ranging from 3 hours to 24 hours. The sample can be whole blood, isolated peripheral blood mononuclear cells (PBMCs) or isolated monocytes. After incubation, the medium in which the cells were incubating is stored and frozen for assay in the same ways as described above.

Molecular indicators of inflammation are increasingly utilized in stress research, including expression of proinflammatory genes and transcription factors that regulate inflammatory activity.  These include nuclear factor kappa B (NF-κB) and activator protein 1 (AP1), among others (Irwin & Cole, 2011).  A variety of chronic stressors have been linked with increased expression of proinflammatory cytokine genes and markers of inflammatory signaling (Irwin & Cole, 2011).  Evaluation of gene expression, or RNA profiling, requires collection of RNA, which can be done with standard venipuncture or DBS. One of the advantages of this approach is that investigators can use bioinformatics techniques to identify transcription factors that may be driving alterations in gene regulation as well as the cellular source of elevated inflammation (Cole et al., 2005).

 

Consideration for Covariates

Like all other biological processes, markers of inflammation are influenced by a variety of factors in addition to stress. O’Connor and colleagues (2009) provide an overview of variables known to be associated with circulating measures of inflammation and, in some cases, stimulated cytokine production. Unless relevant to the study question, researchers should consider requiring that participants refrain from acute exercise, caffeine use, tobacco use, alcohol use and sleep loss for 10-12 hours prior to a fasting blood draw. In addition, the following variables should be assessed and potentially treated as covariates in statistical models: age, sex, socioeconomic status, race/ethnicity, body mass index, alcohol use, sleep behavior, medication use (particularly aspirin, statins, and anti-hypertensives, and antidepressants), and menopausal status (O'Connor et al., 2009). However, covariates should be used thoughtfully and sparingly so that measures across studies are comparable, that is, they use the same covariates and therefore measure the same partial construct (Segerstrom, 2009).

 

Author and Reviewer(s):

This summary was prepared by Dr. Aric Prather, with contributions from Brianna Natale and Dr. Anna Marsland. Reviewed by Drs. Julie Bower, Jan Kiecolt-Glaser, and Suzanne Segerstrom. Please direct general suggestions and feedback to Aric.Prather@ucsf.edu and questions specific to dried blood spot collection to Brianna Natale (BNN11@pitt.edu).

References:

Bosch, J.A., 2014. The use of saliva markers in psychobiology: mechanisms and methods. Diagn. Disord. 24, 99–108.

Cole, S. W., Yan, W., Galic, Z., Arevalo, J., & Zack, J. A. (2005). Expression-based monitoring of transcription factor activity: the TELiS database. Bioinformatics (Oxford, England), 21(6), 803–810. https://doi.org/10.1093/bioinformatics/bti038

 

Fernandez-Botran, R., Miller, J.J., Burns, V.E., Newton, T.L., 2011. Correlations among inflammatory markers in plasma, saliva and oral mucosal transudate in post-menopausal women with past intimate partner violence. Brain, behavior, and immunity 25, 314-321.

Irwin, M. R., & Cole, S. W. (2011). Reciprocal regulation of the neural and innate immune systems. Nature reviews. Immunology, 11(9), 625–632. https://doi.org/10.1038/nri3042

Kiecolt-Glaser, J.K., Preacher, K.J., MacCallum, R.C., Atkinson, C., Malarkey, W.B., Glaser, R., 2003. Chronic stress and age-related increases in the proinflammatory cytokine IL-6. Proceedings of the National Academy of Sciences of the United States of America 100, 9090-9095.

Marsland, A.L., Walsh, C., Lockwood, K., John-Henderson, N.A., 2017. The effects of acute psychological stress on circulating and stimulated inflammatory markers: A systematic review and meta-analysis. Brain, behavior, and immunity.

McDade, T. W., Burhop, J., & Dohnal, J. (2004). High-sensitivity enzyme immunoassay for C-reactive protein in dried blood spots. Clinical chemistry, 50(3), 652-654.

Medzhitov, R., 2008. Origin and physiological roles of inflammation. Nature 454, 428-435.

Meier-Ewert, H.K., Ridker, P.M., Rifai, N., Price, N., Dinges, D.F., Mullington, J.M., 2001. Absence of diurnal variation of C-reactive protein concentrations in healthy human subjects. Clinical chemistry 47, 426-430.

Miller, G., Chen, E., Cole, S.W., 2009. Health psychology: developing biologically plausible models linking the social world and physical health. Annual review of psychology 60, 501-524.

Miller, E. M., & McDade, T. W. (2012). A highly sensitive immunoassay for interleukin‐6 in dried blood spots. American Journal of Human Biology, 24(6), 863-865.