We have found that in pediatric CF, plasma TGF-β1 is increased in association with PsA infection and diminished lung function, and is reduced in association with clinically-effective treatment for a pulmonary exacerbation. Furthermore, our data underscore that TGF-β1 protein measurements are not interchangeable between platelet-free plasma and serum. Specifically, serum TGF-β1 levels were >20-fold higher than plasma levels with no correlation between serum TGF-β1 and either plasma or BALF TGF-β1. In contrast, plasma TGF-β1 was significantly correlated with BALF TGF-β1 with a consistent decrease in plasma TGF-β1 associated with a clinically-meaningful response to therapy. Our data thus support exploration of platelet-free plasma TGF-β1 as a peripheral biomarker of CF lung disease in childhood, and suggest a negative relationship between elevated TGF-β1 protein levels and lung function in pediatric CF lung disease.
When plasma is utilized for studies of TGF-β1
, careful attention must be placed on the method of plasma processing to limit the contribution of activated platelet degranulation ex vivo
. In our experience, important technical steps include prevention of coagulation (immediate processing), collection through a central venous catheter if available, selection of the appropriate specimen tube (citrate preferred as both heparin and EDTA can activate TGF-β1
), adequate centrifugation (to segregate a platelet-free fraction) and use of a sensitive and specific TGF-β1
detection assay. Utilizing these techniques, the plasma levels of TGF-β1
in this study of CF patients were consistent with those reported in the literature from optimally collected plasma specimens10,11,13,26
. Our results prompt critical review of previous literature linking TGF-β1
genotype with TGF-β1
protein measurements in blood. Our data suggest that serum TGF-β1
is of limited relevance to CF lung disease phenotype compared to plasma. Moreover, for studies that evaluate the association of TGF-β1
genotype and plasma TGF-β1
protein, the methods of plasma acquisition and processing should be carefully considered.
While aberrant inflammatory cytokine profiles are well-documented in CF BALF specimens17,27,28,29
, parallel findings of altered cytokine protein levels in the peripheral circulation have been limited. To our knowledge, this is the first study which has demonstrated a significant correlation between plasma and BALF TGF-β1
in the pediatric CF population. Additionally, the correlation between plasma and BALF TGF-β1
in the context of TGF-β1
as disease modifier of CF lung disease severity1,2,4,30
suggest that plasma TGF-β1
may hold promise as a relatively noninvasive biomarker to assess CF lung disease. While this study indicates that plasma TGF-β1
meets the classic definition of a biomarker as an objectively-measureable biologically-relevant characteristic31
, future studies as delineated by Mayer-Hamblett et al32
will be necessary to evaluate its full utility as a CF biomarker. These include assessment of its clinical and therapeutic relevance, sensitivity and specificity across a variety of clinical scenarios, reproducibility within the same patient and across CF centers, and feasibility for wide-spread collection.
The potential utility of plasma TGF-β1 to track CF lung disease is further underscored by the association of increased plasma TGF-β1 with PsA infection and diminished lung function ( and ). While the mechanisms of these associations are beyond the scope of this study, our data point towards relationships between increased TGF-β1 levels and disease severity. It is not clear whether elevated TGF-β1 levels represent a cause or consequence of CF lung disease, but the reduction of TGF-β1 plasma levels in response to treatment may identify a useful biomarker for CF therapeutic interventions, and possibly a target for disease modification.
One important finding of this study was the significant association between an elevated admission plasma TGF-β1
and lung function that remains below normative values despite antibiotic therapy. Increased cytokine measurement during the time of exacerbation has been felt to reflect airway inflammation, and we have previously shown an association between neutrophilic inflammation and BALF TGF-β119
. However, the link between elevated TGF-β1
(a potent pro-fibrotic mediator) at the time of admission and lung function at the conclusion of conventional therapy suggests that TGF-β1
may play a role in pathogenic airway remodeling that is refractory to traditional therapy. Supportive of this hypothesis is data from Hilliard et al29
that reveals a significant association between BALF TGF-β1
and reticular basement membrane thickness demonstrated by endobronchial biopsy. Thus, we speculate that TGF-β1
may bridge the inflammatory and remodeling pathways in CF, serving as one potential mechanism through which TGF-β1
may modify CF lung disease progression.
An important caveat of this study is that we measured and correlated total TGF-β1
protein levels rather than the transient, biologically active form in tissue compartments. Total and active TGF-β1
are not always highly correlated and local activation is a tightly regulated process that independently influences biologic effect33
We additionally recognize the limitation that this study was not designed to identify the sources of plasma TGF-β1
. The positive correlation between both BALF TGF-β1
and platelet count suggests both a pulmonary and platelet contribution to plasma TGF-β1
. While an association is to be expected as platelets are reported to be a major source of circulating TGF-β110
, future studies could consider the use of a specific marker of ex vivo
degranulation such as Platelet Factor 4 (PF4) to insure that measured protein levels represent circulating levels rather than an artifact of specimen processing.
Finally, the relatively small sample size limited our ability to perform multivariable analyses, such as multiple regression analyses with inflammatory cytokines, as well as limiting our ability to form conclusions about TGF-β1
genotype-phenotype relationships. Using an odds ratio for disease of 2.2 and a predicted prevalence of the codon 10 CC genotype of 13–17%2,3
, a study population of several hundred subjects would be necessary to detect significant associations between TGF-β1
genotype, protein concentration and disease severity. Instead, we chose to focus this single-center study on emphasizing the importance of TGF-β1
biospecimen selection and processing by emphasizing relationships with key clinical parameters. Our results help lay the foundation for larger multi-center studies to examine TGF-β1
genotypes, their relationship with TGF-β1
protein levels and clinical outcome measures.
Despite these considerations, our findings suggest that plasma TGF-β1 should be included in future CF biospecimen profiles with further investigation of the mechanisms through which TGF-β1 may modulate CF lung disease progression. The results of this study provide evidence that plasma is the preferred blood biospecimen for measuring TGF-β1 protein levels in CF, as plasma TGF-β1 correlates with BALF levels and fluctuates in association with several important parameters of CF lung disease severity. Whether plasma TGF-β1 is a potentially useful biomarker of disease progression or response to therapy merits further investigation.