Ibuprofen use is associated with reduced C-reactive protein and interleukin-6 levels in chronic spinal cord injury

Abstract

Objective: To assess the association between ibuprofen use and the systemic inflammatory biomarkers C-reactive protein (CRP) and interleukin-6 (IL-6) in chronic Spinal Cord Injury (SCI).

Study design: Prospective cohort study.

Setting: Community dwelling individuals with SCI.

Participants: 338 (278 male, 60 female) community dwelling individuals with chronic SCI (≥1-year post-injury).

Interventions: None.

Main outcome measures: CRP and IL-6 levels were quantified by ultra-sensitive ELISA assay. General linear models were used to assess associations between various clinical and demographic factors and CRP and IL-6 levels.

Results: There were 50 active ibuprofen users and 288 non-users. After adjusting for clinical and demographic factors, ibuprofen users had significantly lower CRP levels (2.3 mg/L versus 3.5 mg/L, P = 0.04) and IL-6 levels (3.2 pg/ml versus 4.0 pg/ml, P = 0.04) compared to nonusers.

Conclusions: Our study suggests that self-reported ibuprofen use may be negatively associated with CRP and IL-6 levels in chronic SCI after adjusting for known confounding factors, and suggests ibuprofen use may be an important, potential variable to consider in future studies focused on systemic inflammation in SCI. Future prospective studies require assessing frequency, duration, and dosage-dependent effects of ibuprofen on systemic markers of inflammation in chronic SCI. These findings may support future clinical trials to determine safety and efficacy of ibuprofen treatment for various outcomes in chronic SCI.

Introduction

Spinal cord injury (SCI) is associated with an ensuing chronic, pro-inflammatory state.^1,2 Inflammation after SCI is not self-limiting, and persistent inflammation may contribute to secondary injury both in the central nervous system (CNS) and systemically.^1,3 Elevated levels of markers of systemic inflammation, including C-reactive protein (CRP) and Interleukin 6 (IL-6), have been demonstrated following SCI.^4–7 A growing body of evidence suggests an inflammatory state contributes to poor outcomes in SCI including increased risk of cardiovascular disease, reduced pulmonary capacity, and immune system dysfunction.^1,4,8–11

The mechanism for this pro-inflammatory state is likely multifactorial including dysfunction of the autonomic nervous system, hypothalamic-pituitary-axis, and metabolic disorder associated with SCI.¹ Within the general population, changes in CRP and/or IL-6 levels are noted with sex, age, race, BMI, and comorbid conditions including heart disease and smoking tobacco.^12–17 Additionally, various clinical contributors including asymptomatic bacteriuria, urinary tract infections (UTI), skin pressure injury, vitamin D levels, and mobility status are associated with elevated CRP and/or IL-6 levels specifically in SCI.^{4,5,11,18–21}

Nonsteroidal anti-inflammatory drugs (NSAIDs) reduce inflammation and produce analgesic effects by suppression of prostaglandins classically via the cyclo-oxygenase (COX) enzyme pathway.^22,23 Recently, novel targets have been identified with NSAID therapy which may modulate inflammation and demonstrate neuroprotective targets in animal models.^24,25 In one human study NSAIDs reduced CRP and IL-6 levels following aneurysmal subarachnoid hemorrhages (SAH).²⁶ However, there are no reports on the effect of NSAID use on systemic inflammation after SCI in humans. In our previous work, we have demonstrated an association between markers of systemic inflammation and pulmonary function. We have also identified factors associated with CRP or IL-6 levels, including ambulatory status, BMI, urinary catheter use, and pressure injury.^8,9,11 However, we have not previously considered the impact of anti-inflammatory drug use on these associations. Thus, the focus of the current study was to assess the association between NSAID use and the systemic inflammatory markers CRP and IL-6, after adjusting for potential confounding factors in men and women with chronic SCI.

Materials and methods

Participants

We studied 338 men and women with chronic SCI enrolled in the Fracture Risk after SCI (FRASCI) observational cohort study. Details regarding eligibility for this study have been previously reported.^27,28 Briefly, inclusion criteria included age 22 years or more and chronic SCI (≥1 year after injury). Exclusion criteria included concomitant neuromuscular disease, tracheostomy, or ventilator-dependent SCI. 348 participants were enrolled and completed testing between August 2009 and December 2014. 10 participants were excluded from the analysis due to missing data (body composition, n = 5 or biomarker results, n = 5) (Fig. 1). All participants provided informed consent and the study was approved by our Institutional Review Board.

Figure 1.

FRASCI inflammation sub-study cohort.

Instruments

Participants completed a questionnaire regarding demographics, medical history, medication use, mobility mode, health habits, current infections/illness, and comorbidities as previously described.²⁹ At the time of study entry, participants were asked to report history of Ibuprofen, Advil^®, or Motrin^® use. If they were actively using one of these medications, duration of use in years was reported. Participants who reported a history of use that was not active at the time of testing were asked to report how long ago (in years) they discontinued use. Ibuprofen users were defined as those actively taking the drug at the time of testing. Nonusers were defined as those never taking the drug or previously taking it but stopping prior to testing. Injury severity was confirmed based on the American Spinal Injury Association Impairment Scale (AIS) and then considered in the following 2 categories: motor complete (AIS A/B) or motor incomplete (AIS C or D). Smoking was defined as smoking 20 or more packs of cigarettes or using 336 g (12 oz) of tobacco or more in a lifetime or smoking 1 or more cigarettes a day for at least 1 year. Current smokers reported cigarette use within one month of testing. Smoking status was considered dichotomously (current smoker versus never/past smoker) and smoking exposure was considered continuously. Primary mode of mobility was considered more than 50% of the time use of a wheelchair or walked with or without assistance. Mobility was considered dichotomously (wheelchair users versus walkers) as in our previous studies.^11,27,28,30 Participants were weighed and length measured in supine for the calculation of body mass index (BMI). In participants with severe joint contractures, length was self-reported (n = 31).

Biochemical analysis

Subjects were asked to undergo testing in a fasting state and efforts were made to collect samples in the morning before a meal. For subject safety, individuals were advised to have a light meal or snack if fasting could worsen a medical condition (orthostatic hypotension). In all cases, information was collected on time since last meal or snack. Plasma samples were drawn into an EDTA tube and stored at −80°C until batch analysis. All biochemical analyses were performed at the Clinical & Epidemiologic Research Laboratory, Department of Laboratory Medicine at Children’s Hospital in Boston, a state-of-the-art reference laboratory that specializes in micro-analysis. High-sensitivity CRP was determined by using a high-sensitivity immunoturbidimetric assay with a sensitivity of .03 mg/L. The day-to-day variability of the assay at concentrations of 0.91, 3.07, and 13.38 mg/L are 2.81, 1.61, and 1.1%, respectively. Interleukin-6 (IL-6) was determined by ultra-sensitive ELISA (R & D Systems, Minneapolis, MN) with a sensitivity of 0.094 pg/ml and day-to-day variability of 9.6, 7.2 and 6.5% at concentrations of 0.49, 2.78 and 5.65 pg/mL, respectively. The assay is linear up to 100 ng/ml, and sensitive to 1 ng/ml. Day-to-day precision (%CV) at various levels of 25OHD ranged from 5.6% to 8.5%. All assays were performed in duplicate and any duplicate with >10% CV was repeated.

Data analysis

All analyses were performed using SAS 9.4 (SAS Institute, Inc., Cary, NC). Neither CRP nor IL-6 were normally distributed and therefore we log transformed each to normalize the distribution of the outcomes. We initially used T-tests or χ² tests to compare subject characteristics as appropriate. General linear models (PROC GLM) were applied to assess associations between various factors and CRP or IL-6 levels. Separate models were constructed for CRP or IL-6. We considered factors that had previously been shown to be associated with either CRP or IL-6 levels, including sex,³¹ age,¹² race,³¹ vitamin D level,^20,21 BMI,⁴ comorbid conditions,^13,14,16 active statin or aspirin medication use,¹⁵ ambulatory status,¹¹ smoking status,¹⁷ and active infections (urinary tract infections and skin pressure injuries).¹¹ For multivariable models, factors with a P value of <0.10 in the univariate models were included in the multivariable models assessing the association between ibuprofen use and CRP or IL-6 levels (PROC GLM). We used backward stepwise elimination of variables which no longer were significant where CRP or IL-6 were considered in separate models as the dependent variables.

Results

Participant characteristics

Demographic information is presented in Table 1. The average age was 54.3 ± 14.3 (SD) years (ranges from 22.8 to 87.6 years) and the average injury duration was 17.4 ± 13.3 years (ranges from 1.0 to 61.1 years). Most were male in sex (82%) and white in ethnicity (87%). 63% of participants used a wheelchair as their primary mode of mobility and 47% had a motor complete injury. The mean BMI was 27.3 ± 6.1 kg/m². 17% of participants were active tobacco smokers, 14% reported diabetes, 37% reported hypertension, and 11% reported a history of heart disease. 11% reported a skin pressure injury and 7% reported an UTI at time of testing. 44% of all participants had a CRP level ≥3 mg/L. Among active ibuprofen users the majority reported ibuprofen use for >1 year (90%). Ibuprofen users had significantly lower mean CRP (3.9 mg/L vs 7.9 mg/L) and IL-6 levels (2.6 mg/L vs 3.9 mg/L) than nonusers (P < 0.01). There were no other significant differences in clinical or demographic characteristics based on ibuprofen use.

Table 1. FRASCI-inflammation cohort participant characteristics.

Variable	Ibuprofen use (n = 50)	No ibuprofen (n = 288)	Total cohort (n = 338)	P
Demographics
Age (years) [Mean ± SD]	53.9 ± 12.3	54.4 ± 14.6	54.3 ± 14.3	0.85
Males, n(%)	44 (88.0)	234 (81.3)	278 (82.3)	0.25
White, n(%)	41 (82.0)	252 (87.5)	293 (86.7)	0.29
Years post injury [Mean ± SD]	16.9 ± 12.2	17.5 ± 13.5	17.4 ± 13.3	0.75
Wheelchair users, n (%)	28 (56.0)	186 (64.6)	214 (63.3)	0.25
Injury completeness
Motor complete
A/B, n(%)	22 (44.0)	136 (47.2)	158 (46.8)	0.67
Motor incomplete
C, n(%)	2 (4.0)	34 (11.8)	36 (10.6)
D, n(%)	26 (52.0)	118 (41.0)	144 (42.6)
Body composition
BMI (kg/m2) [Mean ± SD]	28.5 ± 6.3	27.1 ± 6.1	27.3 ± 6.1	0.15
Health habits
Current smoker, n(%)	9 (18.0)	49 (17.0)	58 (17.2)	0.86
Cigarette exposure (pack-years) [Mean ± SD]	22.9 ± 31.4	27.1 ± 28.6	26.5 ± 29.0	0.47b
Hypertension, n(%)	21 (42.0)	103 (35.8)	124 (36.7)	0.40
Heart disease, n(%)	2 (4.0)	36 (12.5)	38 (11.2)	0.08
Diabetes, n(%)	7 (14.0)	40 (13.9)	47 (13.9)	0.98
Current skin pressure injury, n(%)	3 (6.0)	35 (12.2)	38 (11.2)	0.20
Current UTI, n(%)	2 (4.0)	23 (6.8)	25 (7.4)	0.56
Biomarkers
CRP (mg/L) [Mean ± SD]	3.9 ± 5.3	7.9 ± 16.8	7.3 ± 15.7	0.001a
CRP ≥ 3 (mg/L), n%	17 (34.0)	133 (46.2)	150 (44.4)	0.11
IL-6 (pg/mL) [Mean ± SD]	2.6 ± 2.0	3.9 ± 5.0	3.7 ± 4.7	0.0008

^aObservations available for n = 337; ^bAmong n = 200 ever smokers.

Clinical factors associated with inflammatory biomarkers

Time since last meal or snack was not significantly associated with either CRP or IL-6 (P = 0.95 and P = 0.89, respectively). CRP levels were positively associated with BMI, were lower in ibuprofen users, white individuals compared to other races, walkers compared to wheelchair users, motor incomplete SCI compared to motor complete SCI, and greater in individuals with current skin pressure injury and UTI (Table 2(a)). In multivariable models adjusting for these factors (Table 3(a)), CRP levels were no longer significantly associated with current UTI (P = 0.43). CRP levels increased 1.1 mg/L for every unit increase in BMI, were greater in wheelchair users, lower in white individuals compared to others, and greater in participants with current skin pressure injury. When adjusting for these factors, active ibuprofen users had significantly lower CRP levels than non-user (2.3 mg/L vs. 3.5 mg/L P = 0.04). Results were similar when considering motor completeness instead of walking status in this model.

Table 2. (a) Univariate factors associated with natural log CRP.

Continuous variables	β	eβ	P
Age (years)	–0.0002	0.99	0.97
Years post injury	–0.002	0.99	0.74
BMI (kg/m2)	0.071	1.07	<0.0001
25OH Vitamin D (ng/ml)	–0.007	0.99	0.28
Cigarette exposure (pack-years)	0.004	1.00	0.23a
Categorical variables	Mean ln CRP	e(ln CRP) (mg/L)	P
Sex
Male	0.991	2.693	0.36
Female	0.807	2.241
Race
White	0.897	2.452	0.04
Other	1.353	3.869
Walking status
Wheelchair user	1.226	3.407	<0.0001
Walk with or without aid	0.499	1.647
Injury completeness
Motor complete	1.204	3.333	0.003
Motor incomplete	0.744	2.104
Current smoker
Yes	1.082	2.951	0.47
No	0.933	2.542
Hypertension
Yes	1.046	2.846	0.39
No	0.907	2.476
Heart disease
Yes	1.176	3.241	0.32
No	0.931	2.537
Diabetes
Yes	0.989	2.688	0.87
No	0.953	2.593
Current skin pressure injury
Yes	1.682	5.376	0.0008
No	0.867	2.379
Current UTI
Yes	1.596	4.933	0.02
No	0.907	2.476
Ibuprofen use
Yes	0.648	1.911	0.09
No	1.012	2.751
Statin use
Yes	0.799	2.223	0.16
No	1.032	2.806
Aspirin use
Yes	1.058	2.880	0.46
No	0.927	2.526
aAmong n = 200 ever smokers.
β – standardized coefficients.

Table 2 (b) Univariate factors associated with natural log IL-6.
Continuous variable	β	eβ	P
Age (years)	0.013	1.01	<0.0001
Years post injury	0.004	1.00	0.30
BMI (kg/m2)	0.053	1.05	<0.0001
25OH Vitamin D (ng/ml)	−0.005	0.99	0.25
Cigarette exposure (pack-years)	0.002	1.00	0.31a
Categorical variables	Mean ln CRP	e(ln CRP) (mg/L)	P
Sex
Male	0.925	2.521	0.16
Female	0.751	2.119
Race
White	0.888	2.430	0.73
Other	0.937	2.552
Walking status
Wheelchair user	0.972	2.643	0.03
Walk with or without aid	0.761	2.140
Injury completeness
Motor complete	0.921	2.511	0.60
Motor incomplete	0.871	2.389
Current smoker
Yes	1.117	3.056	0.03
No	0.848	2.335
Hypertension
Yes	1.045	2.843	0.02
No	0.807	2.241
Heart disease
Yes	1.248	3.483	0.008
No	0.849	2.337
Diabetes
Yes	1.292	3.640	0.0007
No	0.829	2.291
Current skin pressure injury
Yes	1.457	4.293	<0.0001
No	0.823	2.277
Current UTI
Yes	1.257	3.514	0.03
No	0.865	2.375
Ibuprofen use
Yes	0.684	1.981	0.07
No	0.930	2.534
Statin use
Yes	1.040	2.829	0.04
No	0.828	2.288
Aspirin use
Yes	1.176	3.241	0.0007
No	0.803	2.232

^Aamong n = 200 ever smokers.

β – standardized coefficients.

Table 3. (a) Multivariable factors associated with natural log CRP.

ln CRP, <0.0001 r2 = 0.22
Continuous variable	β	eβ	P
BMI (kg/m2)	0.081	1.084	<0.0001
Categorical variable	Mean ln CRP	e(ln CRP) (mg/L)	P
Walking status
Wheelchair user	1.454	4.280	<0.0001
Walk with or without aid	0.645	1.905
Race
White	0.851	2.341	0.05
Other	1.248	3.483
Current skin pressure injury
Yes	1.296	3.654	0.02
No	0.803	2.232
Ibuprofen use
Yes	0.852	2.344	0.04
No	1.247	3.479
β – standardized coefficient.

Table 3 (b) Multivariable factors associated with natural log IL-6.
ln IL-6, <0.0001 r2 = 0.32
Continuous variable	β	eβ	P
Age (years)	0.013	1.013	<0.0001
BMI (kg/m2)	0.058	1.059	<0.0001
Categorical variable	Mean ln IL-6	e(ln IL-6) (pg/mL)	P
Walking status
Wheelchair user	1.478	4.384	<0.0001
Walk with or without aid	1.050	2.857
Current smoker
Yes	1.504	4.499	<0.0001
No	1.024	2.784
Heart disease
Yes	1.430	4.178	0.01
No	1.099	3.001
Current skin pressure injury
Yes	1.548	4.702	<0.0001
No	0.981	2.667
Ibuprofen use
Yes	1.153	3.167	0.04
No	1.376	3.959

β – standardized coefficient.

In univariate analyses IL-6 levels were positively associated with age, BMI, were greater in wheelchair users compared to walkers, current smokers, history of hypertension, heart disease, diabetes, current skin pressure injury, current UTI, active statin use, active aspirin use, and lower in active ibuprofen users (Table 2(b)). In multivariable models adjusting for these factors (Table 3(b)), IL-6 levels were no longer significantly associated with history of hypertension, diabetes, current UTI, statin use, or aspirin use (P = 0.19–0.78). IL-6 levels increased 1.01 pg/ml for every year increase in age. IL-6 levels increased 1.059 pg/ml for every unit increase in BMI. IL-6 levels were greater in wheelchair users, current smokers, participants with heart disease, and participants with active skin pressure injuries. After adjusting for these factors, active ibuprofen users had significantly lower IL-6 levels than non-users (3.2 pg/ml versus 4.0 pg/ml, P = 0.04). Results were similar when considering motor completeness instead of walking status in this model.

Discussion

We examined circulating markers of systemic inflammation (CRP and IL-6) in the setting of active ibuprofen use in 338 community dwelling men and women with chronic SCI. IL-6 and CRP levels both increased with BMI and were higher in wheelchair users compared to walkers and those with active skin pressure injuries. IL-6 was additionally increased with age, history of heart disease, and active smokers, while CRP was also decreased in white individuals compared to other races. When adjusting for these factors, active ibuprofen use was significantly associated with lower levels of both CRP and IL-6. These associations were independent of active infection, skin pressure injury, comorbidities, or other previously reported factors associated with CRP or IL-6 levels in SCI.^5,6,9,11

In this study we identified several factors associated with both CRP and IL-6. These findings are consistent with previous findings that IL-6 regulates CRP production. CRP belongs to a group of serum proteins described as acute phase reactants, mostly produced by hepatocytes, that are activated by noxious stimuli such as infection or tissue injury. Specifically, CRP and serum amyloid A are the two most potently activated acute phase reactants in humans.³² CRP exponentially rises after noxious stimuli and is a clinically useful marker for monitoring the course of diseases, infections, and chronic inflammation.³³ IL-6 is a cytokine released from surrounding endothelial cells, fibroblasts, and leukocytes as part of the local reaction to a noxious stimulus. IL-6 is one of many cytokines that activates the systemic reaction including acute phase reactants, and has been demonstrated in transgenic mouse and human hepatocyte cell line models to be necessary for the expression of CRP.^32,34,35 IL-6 is also produced by myocytes and adipocytes and has been demonstrated to regulate systemic metabolism, bone homeostasis, as well as to modulate pain responses.^36,37

To our knowledge, this is the first report that active ibuprofen use is associated with significantly lower levels of systemic inflammatory biomarkers in chronic SCI. Our findings are consistent with a previous study demonstrating that ibuprofen use was associated with lower CRP or IL-6 levels in 138 patients with SAH.²⁶ This has potential clinical relevance given that elevated IL-6 levels were associated with a four-fold increase in risk of unfavorable outcomes measured by the Glasgow coma scale after SAH.³⁸ In SCI chronic systemic inflammation is thought to be multifactorial and previously conceptualized as a consequence of reduced mobility, body composition changes, and frequency of infections.^11,39 Many studies have established the relationship of chronic inflammation and several detrimental health and functional outcomes including immune system dysfunction, reduction in pulmonary function, and increased chronic neuropathic pain specifically in the SCI population.^1,9,39 Similarly, in the general population, elevated CRP levels are associated with increased cardiovascular risk with CRP >3 mg/L defined as high risk.^13,40 CRP levels were a better predictor of cardiovascular events than cholesterol levels or known risk factors alone in a large prospective trial in women in the general population.¹³ This is of interest as SCI is associated with increased cardiovascular disease compared to the general population.⁴¹ Our study demonstrated an overall mean CRP of 7.3 ± 15.7 mg/L, more than double the high-risk category cut-point. Interestingly, the active ibuprofen users had lower CRP levels, although the mean was still greater than >3 mg/L threshold of the high-risk category.

There is emerging consensus that inflammation contributes to negative health and functional outcomes in both the general population and after neurotrauma. It remains unknown if therapeutic approaches to reduce inflammation improve outcomes. Statin medications reportedly reduce systemic inflammation independent of their effect on cholesterol. Rosuvastatin treatment significantly reduced major cardiac events in healthy men and women with elevated CRP but without hyperlipidemia ¹⁵. In animal models of SCI, statins have previously been associated with improvements in motor recovery and reduced markers of inflammation, including IL-6.⁴² While NSAIDs are also known to reduce inflammation, there is controversy regarding their impact on both cardiovascular health and overall safety profile.^43,44 The long-term effect of chronic NSAID use on cardiovascular health has not been studied in the SCI population.

NSAIDs classically modulate inflammation by suppression of prostaglandin production via the COX enzyme pathway.⁴⁵ NSAIDs regulate inflammation via additional pathways including acting as proliferator-activated receptor gamma (PPARγ) agonists and modulation of NFkB and its downstream targets.^22,23,46 Another pathway of particular interest that has been associated with reduced neuroinflammation is the inhibition of GTPase Rho by NSAIDS.^25,47 Preclinical research has demonstrated activation of Rho GTPase as a converging pathway which leads to inhibition of axonal regeneration in central nervous system injuries.^48,49 A meta-analysis of animal models of SCI have additionally demonstrated treatment with ibuprofen or indomethacin to have overall improvements in motor scores, and Rho inhibitors are targets of multiple clinical trials for improvements in motor recovery after SCI.^25,50 It is possible, therefore, that NSAID use may be neuroprotective in SCI and other forms of neurotrauma.

Limitations

This study has several limitations to consider. Our participants were community dwelling individuals with injury duration of 1 year or more. Therefore, it does not address the potential associations between ibuprofen use and inflammatory markers in the acute setting of SCI. Our participants race was 85% white. This is not representative of the current (2015–2020) U.S. population of those living with SCI with 58.9% being non-Hispanic White.⁵¹ This affects the generalizability of the study. Primary mode of mobility was considered more than 50% of the time. Our “majority” dichotomous mobility definition cannot assess an individual living with a SCI’s physical activity, independence, and variability in modes of mobility, however we previously validated this definition in detecting an association with CRP levels in SCI in previous and current study cohorts.^11,27,28,30 While our findings suggest that ibuprofen use may reduce systemic inflammatory mediators and potentially have positive health benefits among those living with a chronic SCI, additional work is needed to confirm these findings in prospective clinical trials. It is important to note that activation of various anti-inflammatory pathways is heterogenous and dependent of type of NSAIDs as well as dosage and duration of use. For example, ibuprofen has demonstrated strong Rho-inhibition in a dose dependent manner while naproxen does not.^47,52 The majority of ibuprofen users in our study where chronic users greater than 1 year. Use was self-reported, and we lacked the ability to analyze biomarker variation based on dosage or duration of use with greater specificity. These associations warrant further investigation in future studies.

Conclusion

The chronic inflammatory state which is frequently seen after SCI leads to poor health and functional outcomes. We report that self-reported ibuprofen use is negatively associated with systemic markers of inflammation, specifically CRP and IL-6, in chronic SCI, and suggests ibuprofen use may be an important, potential variable to consider in future studies focused on systemic inflammation in SCI. Future prospective studies require assessing frequency, duration, and dosage-dependent effects of ibuprofen on systemic markers of inflammation in chronic SCI. These findings may support future clinical trials to determine safety and efficacy of ibuprofen treatment for various outcomes in chronic SCI.

Funding Statement

This study was supported by The National Institute of Arthritis and Musculoskeletal and Skin Diseases under grants [1R01AR059270-01; R01AR064793].

Disclaimer statements

Contributors None

Declaration of interest Andrew Park, Dustin Anderson, Leslie Morse, Nguyen Nguyen, and Ricardo Battaglino declare that they have no conflict of interest.

Conflicts of interest Authors have no conflict of interests to declare.

Data availability statement

The data that support the findings of this study are available from the corresponding author, LM, upon reasonable request