Serum galectins as potential biomarkers of inflammatory bowel diseases.

The inflammatory bowel diseases (IBD), which include mainly Crohn's disease (CD) and ulcerative colitis (UC), are common chronic inflammatory conditions of the digestive system. The diagnosis of IBD relies on the use of a combination of factors including symptoms, endoscopy and levels of serum proteins such as C-reactive protein (CRP) or faecal calprotectin. Currently there is no single reliable biomarker to determine IBD. Galectins are a family of galactoside-binding proteins that are commonly altered in the circulation of disease conditions such as cancer and inflammation. This study investigated serum galectin levels as possible biomarkers in determining IBD and IBD disease activity. Levels of galectins-1, -2, -3, -4, -7 and -8 were analysed in 208 samples from ambulant IBD patients (97 CD, 71 UC) patients and 40 from healthy people. Disease activity was assessed using Harvey-Bradshaw Index for CD and simple clinical colitis activity index for UC. The relationship of each galectin in determining IBD and IBD disease activity were analysed and compared with current IBD biomarker CRP. It was found that serum level of galectin-1 and -3, but not galectins-2, -4, -7 and -8, were significantly higher in IBD patients than in healthy people. At cut-off of 4.1ng/ml, galectin-1 differentiated IBD from healthy controls with 71% sensitivity and 87% specificity. At cut-off of 38.5ng/ml, galectin-3 separated IBD from healthy controls with 53% sensitivity and 87% specificity. None of the galectins however were able to distinguish active disease from remission in UC or CD. Thus, levels of galectins-1 and -3 are significantly elevated in both UC and CD patients compared to healthy people. Although the increased galectin levels are not able to separate active and inactive UC and CD, they may have the potential to be developed as useful biomarkers for IBD diagnosis either alone or in combination with other biomarkers.

Introduction

Inflammatory bowel diseases (IBD) are common chronic inflammatory conditions of the digestive system. IBD mainly include Crohn’s disease (CD), which can affect any part of the digestive tract, and ulcerative colitis (UC), which affects only the colon[1]. Both CD and UC typically cause symptoms of diarrhoea, abdominal pain, fatigue and weight loss[1]. The prevalence of IBD in Western countries is high and a recent Scottish study estimated a prevalence of 784/ 100, 000 population[2]. The diagnosis of IBD usually relies on a combination of symptoms, laboratory tests such as C-reactive protein (CRP), faecal calprotectin (FC) and endoscopic evaluation. Endoscopic evaluation is considered the gold standard and at present, there are no simple blood markers which accurately diagnose IBD[3].

IBD typically follow a relapsing-remitting pattern with periods of disease activity interspersed with quiescent disease[1]. IBD disease activity in clinical practice is often assessed in a composite manner by (i) evaluation of symptoms using validated indices such as the Crohn’s disease activity index (CDAI) or the Harvey-Bradshaw index (HBI) for CD, and the simple clinical colitis activity index for UC; (ii) assessment of biomarkers such as C-reactive protein (CRP) or faecal calprotectin (FC) and (iii) endoscopic evaluation[3]. Endoscopic assessment is considered the gold standard for assessment of disease activity but repeated endoscopy is impractical, expensive and associated with poor patient acceptance and a potential for complications. CRP is currently the most used non-invasive serum IBD biomarker in clinic. However, CRP determination [4] is limited by poor specificity and sensitivity as (i) CRP level is altered in other acute inflammatory conditions in addition to CD and UC and (ii) up to 25% of patients, who have demonstrable disease activity determined by endoscopy have normal CRP values[5]. FC determination has far greater accuracy for detecting intestinal inflammation than CRP[6] but it requires collection of faecal sample which is cumbersome and has poor patient acceptability[7]. Thus, there is a need for more accurate and non-invasive biomarkers for establishing disease activity in IBD.

Galectins are a family of 15 (so far) mammalian galactoside-binding proteins that share a consensus amino acid sequence in their carbohydrate recognition domains (CRDs)[8]. Based on their structural differences, galectin family members are classified into three subgroups of proto-, chimeric- and tandem-repeat-types. The prototype galectins include galectins-1, 2, 5, 7, 10, 11, 13, 14 and 15, each containing a single CRD in the polypeptide sequences and often identified as non-covalently linked homodimer. The chimera-type galectin, which has only one member galectin-3, is composed of an unstructured non-lectin domain linked to a CRD and can form as multiple homotypic forms such as a pentamer. The tandem-repeat-type galectins include galectin-4, 6, 8, 9 and 12, each containing two different CRDs close to each end of the single polypeptide chain[9].

Galectins are widely expressed by human cells, in particular epithelial and immune cells. Several galectin members including galectins-1, -2, -3, -4 and -8 [10] are known to be expressed by the human intestinal epithelium[11, 12]. A large body of evidence have reported important roles of these galectins in colorectal cancer development, progression and metastasis (e.g. tumor transformation, cancer cell adhesion, invasion, migration and angiogenesis) by galectin interaction with a number of galactose-terminated glycans[13]. Serum levels of galectins such as galectins-2,-3, -4 and -8 are significantly higher in colon cancer patients than in healthy people [14] and promotes circulating tumour cell haematogenous dissemination in metastasis[14-17]. Interactions of galectins with galactose-terminated glycans on cell surface have also been implicated in the pathogenesis of IBD by induction of T cell apoptosis and NF-kappa B signaling [18-20], [19, 21]. Level of serum galectin-3 was reported to be higher in IBD patients compared to healthy controls[22]. However, it is not known whether levels of any other galectin members are also altered in the circulation of IBD patients and whether serum galectins could be used as potential biomarkers for determining IBD and disease activity.

In this study, we compared the serum levels of galectins-1, -2–3, -4, -7 and -8 in patients with UC, CD and healthy people and analyzed the relationship of galectin levels with disease activity in IBD patients.

Materials and methods

Materials

Recombinant human galectins-1, -2, -3, -4, -7 and -8, antibodies against human galectins-1, -2, -3, -4, -7 and -8 and biotinylated antibodies against human galectins-1, -2, -3, -4, -7 and -8 were obtained from R&D Systems (Abingdon, UK). ExtrAvidin peroxidase, FAST OPD and bovine serum albumin were from Sigma-Aldrich (Dorset, UK).

Serum samples

Serum samples were obtained from a cross-sectional study of ambulant patients attending a specialist IBD clinic at the Royal Liverpool University Hospital. A total of 168 samples from IBD (including 97 from CD and 71 from UC) patients and 40 from healthy people were obtained. Ethical approval was obtained from the Liverpool Regional Ethics Committee. Written informed consent was obtained from all participants and the study population was comprised of adults over the age of 18. The diagnosis of IBD was established using conventional clinical, endoscopic and radiological criteria. All patients were eligible for inclusion excepting patients with an ostomy. Baseline details including disease phenotype, disease duration, smoking status, concurrent medications, prior surgical history and HBI for Crohn’s disease and SCCAI for ulcerative colitis were collected on the day of enrolment. The HBI is a 5-item index used to assess disease activity in CD with a score ≤4 indicating remission, scores of 5–7, 8–16 and >16 indicating mild, moderate and severe disease[23]. The SCCAI includes 6 variables with a score of ≤2 indicating remission, scores of 3–5, 6–11 and ≥12 indicating mild, moderate and severe disease[24]. Laboratory tests were conducted as part of routine clinical care and included full blood count, renal and liver function tests, C-reactive protein (CRP) measurement. We investigated the ability of CRP to distinguish active disease from remission using a threshold of CRP≥5mg/L, as described previously [25].

Determination of serum levels of galectins

Serum levels of galectins-1, -2, -3, -4, -7 and -8, to which adequate capture and detection antibodies and recombinant galectins are available commercially for sandwich galectin ELISA, were determined by sandwich ELISA as described in our previous study[14]. Briefly, high-binding 96-well plates were coated with anti-galectin antibody at 2.5 μg/mL in coating buffer (15 mM Na2CO3 and 17 mM NaHCO3, pH9.6) overnight at 4°C. The plate was washed with washing buffer (0.05% Tween-20 in PBS) and incubated with blocking buffer (1% bovine serum albumin in PBS) for 1 hr at room temperature. Serum samples (1:2 or 1:5 dilution in PBS) or standard recombinant galectins were introduced to the plates for 2 hrs. After two wishes with PBS, biotinylated anti-galectin antibody (1.25 μg/mL in blocking buffer) was applied for 1 hr at room temperature before application of ExtrAvidin peroxidase (1:10,000 dilution in blocking buffer) for 1 hour. Following two washes with PBS, the plates were developed with SigmaFAST OPD for 10 minutes. The reaction was stopped by adding 4 mol/L sulfuric acid before the absorbance was read at 492 nm by a microplate reader.

Statistical analysis

Differences between the levels of each galectin in UC, CD and healthy people were analysed by Mann-Whitney U test. Patient demographics are summarised as mean (SD) or median (IQR) for continuous variables and frequency (%) for categorical variables. Mean and median serum galectin levels were calculated to account for inter-individual variability in serum galectin levels. The difference between remission and active disease groups was analysed using Mann-Whitney test. Receiver operating characteristic (ROC) curves were constructed to assess the independent ability of galectins to discriminate between patients with and without clinical remission, using the area under the ROC curve (AUROC). The optimal cut-off point was calculated by minimising the distance between the ROC curve and the upper left hand corner of the plot. Analysis was conducted for all patients combined and then by diagnosis. Similar analyses were conducted to examine the correlation between CRP and disease activity. All analysis was performed using Stata v13 software (State Statistical Software: Release 12. College Station, TX: StataCorp LP), p< 0.05 is considered significant in all cases.

Results

A total of 208 serum samples (40 from healthy people, 97 from CD and 71 from UC patients) were included in the study. The baseline characteristics of the included IBD patients and healthy people are summarised in Table 1. Of the IBD patients, 70 (72.2%) CD patients and 30 (43.3%) UC patients were in clinical remission as defined by HBI≤4 or SCCAI≤2.

Table 1

Baseline characteristics of the IBD patients and control healthy people.

	Controls (n = 40)	Crohn’s disease (n = 97)	Ulcerative colitis (n = 71)
Age, mean (SD), years	48.8 (15.6)	40.9 (15.0)	45.1 (18.7)
Sex     Male	16 (40.0)	36 (37.1)	33 (46.5)
Female	21 (52.5)	61 (62.9)	38 (53.5)
Smoking    Current	5 (12.5)	25 (25.8)	12 (16.9)
Ex	8 (20.0)	18 (18.6)	18 (25.4)
Never	11 (27.5)	54 (55.7)	41 (57.8)
Missing	16 (40.0)
Disease location		Colonic: 26 (26.8)	Proctitis: 14 (19.7)
		Ileal: 34 (35.1)	Left-sided: 37 (52.1)
		Ileocolonic: 27(27.8)	Pancolitis: 20 (28.2)
		Perianal: 25 (25.8%)
Disease duration (years) <1		8 (8.3)	7 (9.9)
1 to 5		15 (15.5)	22 (31.0)
5 to 10		18 (18.6)	40 (56.3)
>10		49 (50.5)	0 (0)
Missing		7 (7.2)	2 (2.8)
Previous surgery		35 (36.1)	0
Medications    5-ASA		26 (26.8)	58 (81.7)
Thiopurines		45 (46.4)	19 (26.8)
Methotrexate		2 (2.1)	1 (1.4)
Anti-TNF agent		19 (19.6)	5 (7.0)
Corticosteroids		6 (6.2)	9 (12.7)
Disease activity   Active		27 (27.8)	41 (57.8)
Remission		70 (72.2)	30 (42.3)
C-reactive protein (mg/L)
>5		29 (29.9)	21 (29.6)
≤5	17 (100)	51 (52.6)	45 (63.4)
Missing		17 (17.5)	5 (7.0)

Categorical variables: values are number (%)

Serum galectin levels in healthy people and IBD patients

Serum levels of six galectin members were analysed. Among these galectin members, two galectins, galectin- 1 and -3 were found to have significantly higher levels in IBD patients compared to the healthy controls. When galectin levels were compared separately between UC and CD with healthy controls, significantly higher levels of galectin-1 (p = 0.0001 and 0.0003) and galectin 3 (p = 0.0003 and 0.0001, respectively) were found in UC and CD respectively (Table 2). Serum levels of galectin-2, -4, -7 and -8 showed no significant differences in UC and CD than in the healthy controls, although their levels in IBD patients were numerically higher. We next analysed the optimal concentration of galectins-1 and -3 that could distinguish IBD from healthy controls. At a cut-off of 4.1ng/ml, galectin-1 differentiated IBD from healthy controls with 71% sensitivity and 87% specificity. At a cut-off of 38.5ng/ml, galectin-3 differentiated IBD from healthy controls with 53% sensitivity and 87% specificity. Finally, we analysed serum levels of galectins-1, -2, -3, -4, -7, and -8 according to disease location (S1 File: S1-S6 Tables), disease duration (S1 File: S7-S12 Tables) and concomitant medications (S1 File: S13-S18 Tables). No significant differences in median galectin values across the variables was observed in any galectin member (S1 File: S1 to S18 Tables). As the sample size in each sub-group in this study was small, future studies with larger sample numbers would help to further clarify this conclusion.

Table 2

Galectin levels in healthy controls, UC and CD patients.

	Controls(n = 40)	Crohn’s disease (n = 97)	Ulcerative colitis (n = 71)	All IBD(n = 168)
Gal-1
N	15	81	61	142
Mean (SD), ng/ml	6.2 (14.2)	42.8 (194.8)	51.7 (154.1)	46.6 (177.9)
Median (range), ng/ml	0.7 (0, 48.1)	7.5 (0.3, 1300)	11.9 (0, 815.9)	9.2 (0,1300)
P value		0.0001	0.0002	0.0001
Gal-2
N	20	87	66	153
Mean (SD), ng/ml	177.7 (616.5)	1068.2 (6229.7)	2628.6 (12845.9)	1741.3 (9650.1)
Median (range)	0.6 (0, 2669.3)	0.1 (0, 56371.4)	0 (0, 99546.4)	0.05 (0, 99546.4)
P value		0.6517	0.7962	0.7163
Gal-3
N	23	96	70	166
Mean (SD), ng/ml	22.0 (22.1)	49.1 (38.8)	55.3 (53.5)	51.7 (45.5)
Median (range), ng/ml	14.9 (0, 96.4)	42.1 (0, 282.1)	37.4 (0.7, 276.2)	41.7 (0, 282.1)
P value		0.0003	0.0001	<0.0001
Gal-4
N	23	88	71	159
Mean (SD), ng/ml	35.7 (58.0)	45.2 (122.6)	180.0 (989.7)	105.4 (668.4)
Median (range),ng/ml	0 (0, 185.5)	0 (0, 868)	0 (0, 8015)	0 (0, 8015)
P value		0.9072	0.8028	0.8403
Gal-7
N	14	70	55	125
Mean (SD), ng/ml	132.2 (163.6)	59.2 (138.7)	52.4 (154.8)	56.2 (145.4)
Median (range), ng/ml	60.1 (0, 466.3)	0 (0, 764.8)	0 (0, 859.7)	0 (0, 859.7)
P value		0.0350	0.1205	0.0574
Gal-8
N	18	93	69	162
Mean (SD), ng/ml	19.4 (31.0)	48.3 (96.6)	100.7 (313.5)	70.6 (218.0)
Median (range), ng/ml	4.7 (0, 104.2)	3.5 (0, 578.5)	18.1 (0, 2549.0)	7.3 (0, 2549.0)
P value		0.103	0.1607	0.4197

Serum galectins in active and inactive IBD

We next analysed the relationship of levels of galectin members in active and inactive CD and UC. None of the serum galectins showed significantly different in active UC and CD group compared to UC (Table 3) and CD (Table 4) groups in remission. When serum CRP levels were examined, significantly higher CRP was seen in patients with both active UC (P = 0.047, Table 3) and CD (P = 0.047, Table 4) compared to patients in remission. We also analysed serum galectin levels according to the clinical severity of disease activity (remission, mild, moderate or severe). We did not identify an association between serum galectin levels and increasing disease severity (S1 File: S19-S24 Tables).

Table 3

Galectin levels in patients with active and inactive UC.

	UC only			P value
	SCCAI ≤2	SCCAI >2	All
Galectin 1
N	25	36	61
Mean (SD), ng/ml	104.9 (232.5)	14.8 (14.3)	51.7 (154.1)
Median (range), ng/ml	13.1 (0.2, 815.9)	10.5 (0, 48.9)	11.9 (0, 815.9)	0.4545
Galectin 2
N	25	41	66
Mean (SD), ng/ml	2078.7 (6474.6)	2964.0 (15578.7)	2628.6 (12845.9)
Median (range), ng/ml	0.1 (0, 29589.9)	0 (0, 99546.4)	0 (0, 99546.4)	0.3704
Galectin 3
N	30	40	70
Mean (SD), ng/ml	46.3 (43.1)	62.0 (60.0)	55.3 (53.5)
Median (range), ng/ml	28.3 (7.2, 186.4)	47.1 (0.7, 276.2)	37.4 (0.7, 276.2)	0.2353
Galectin 4
N	30	41	71
Mean (SD), ng/ml	122.4 (457.6)	222.1 (1248.2)	180.0 (989.7)
Median (range), ng/ml	0 (0, 2494.7)	0 (0, 8015)	0 (0, 8015)	0.4965
Galectin 7
N	21	34	55
Mean (SD), ng/ml	97.7 (226.6)	24.4 (77.4)	52.4 (154.8)
Median (range), ng/ml	0 (0, 859.7)	0 (0, 439.4)	0 (0, 859.7)	0.1672
Galectin 8
N	29	40	69
Mean (SD), ng/ml	134.7 (468.5)	76.1 (110.9)	100.7 (313.5)
Median (range), ng/ml	19.7 (0, 2549.0)	13.5 (0, 403.5)	18.1 (0, 2549.0)	0.6183
CRP
N	28	38	66
Mean (SD), mg/L	4.8 (14.5)	7.3 (12.5)	6.3 (13.3)
Median (range), mg/L	0 (0, 56)	0 (0, 55)	0 (0, 56)	0.0470

Table 4

Galectin levels in patients with active and inactive CD.

	CD only			P value
	HBI ≤5	HBI >5	All*
Galectin 1
N	60	20	81
Mean (SD), ng/ml	52.6 (225.7)	15.1 (18.8)	42.8 (194.8)
Median (range), ng/ml	7.5 (0.3, 1300)	9.4 (0.6, 66.0)	7.5 (0.3, 1300)	0.5938
Galectin 2
N	64	22	87
Mean (SD), ng/ml	1336.6 (7226.3)	336.0 (1198.4)	1068.2 (6229.7)
Median (range), ng/ml	0.2 (0, 56371.4)	0.1 (0, 5545.4)	0.1 (0, 56371.4)	0.6338
Galectin 3
N	69	26	96
Mean (SD), ng/ml	46.4 (41.5)	54.0 (29.2)	49.1 (38.8)
Median (range), ng/ml	40.1 (0, 282.1)	53.4 (12.8, 118.1)	42.1 (0, 282.1)	0.1036
Galectin 4
N	66	21	88
Mean (SD), ng/ml	52.0 (133.0)	25.8 (85.2)	45.2 (122.6)
Median (range), ng/ml	0 (0, 868)	0 (0, 390.5)	0 (0, 868)	0.3931
Galectin 7
N	53	16	70
Mean (SD), ng/ml	60.8 (148.5)	57.8 (108.3)	59.2 (138.7)
Median (range), ng/ml	0 (0, 764.8)	0 (0, 419.6)	0 (0, 764.8)	0.8517
Galectin 8
N	68	24	93
Mean (SD), ng/ml	52.2 (102.4)	39.2 (81.0)	48.3 (96.6)
Median (range), ng/ml	4.0 (0, 578.5)	0 (0, 287.1)	3.5 (0, 578.5)	0.2921
CRP
N	59	21	80
Mean (SD), mg/L	4.6 (8.2)	10.2 (12.1)	6.1 (9.6)
Median (range), mg/L	0 (0, 35)	6 (0, 38)	0 (0, 38)	0.0472

* one patient whose disease activity could not be accurately established was also included in this analysis.

We then analysed if a combination of CRP with serum galectin-1 or -3 was better than either marker on its own in determining disease activity in UC and CD (Table 5). CRP in combination with either galectin-3 was able to distinguish active disease from remission in both UC (P = 0.0219) and CD (0.0056). However, the combination of galectin-3 with CRP was no better than CRP alone in distinguishing active disease from remission (p = 0.4150 from chi-square test comparing AUCs). In contrast to galectin-3, a combination of galectin-1 and CRP was unable to distinguish active disease from remission in both UC (P = 0.39) and UC ((P = 0.11). We then computed receiver operating characteristic (ROC) analysis of CRP alone or in a combination of galectins-1 and -3 and CRP in active and inactive UC and CD (Table 6). This combination did not show any significant improvement than CRP alone in distinguishing active from inactive disease.

Table 5

Combination of galectins-1 and -3 and CRP in active and inactive UC and CD.

	UC only			P value
	SCCAI ≤2	SCCAI >2	All
Galectin 1+CRP
N	23	33	56
Mean (SD)	119.5 (246.3)	21.0 (21.1)	61.4 (164.1)
Median (range),	17.5 (0.2, 822.1)	16.1 (0, 82.3)	16.5 (0, 822.1)	0.3910
Galectin 3+CRP
N	28	37	65
Mean (SD)	45.1 (44.0)	63.1 (47.2)	55.4 (46.3)
Median (range)	28.3 (7.2, 219.8)	58.0 (8.2, 233.7)	36.2 (7.2, 233.7)	0.0219
	CD only			P value
	HBI ≤5	HBI >5	All
Galectin 1+CRP
N	50	15	65
Mean (SD)	66.4 (246.6)	30.4 (24.9)	58.1 (216.6)
Median (range)	14.6 (0.3, 1308)	23 (0.6, 80.3)	15.4 (0.3, 1308)	0.1176
Galectin 3+CRP
N	59	21	80
Mean (SD)	46.3 (42.4)	62.0 (28.1)	50.4 (39.6)
Median (range)	41.4 (0, 297.1)	55.3 (20.6, 124.1)	46.8 (0, 297.1)	0.0056

Table 6

ROC analysis of CRP alone or a combination of galectin 3 and CRP in active and inactive disease.

	UC only	CD only	UC + CD
CRP	0.645 (0.540, 0.749) [N = 65]	0.642 (0.519, 0.765) [N = 80]	0.621 (0.542, 0.700) [N = 145]
Galectin 3+CRP	0.667 (0.529, 0.805) [N = 65]	0.705 (0.579, 0.830) [N = 80]	0.668 (0.579, 0.756) [N = 145]
Galectin 3+CRP (< = />52)	0.637 (0.517, 0.756) [N65]	0.698 (0.580, 0.816) [80]	0.667 (0.588, 0.746) [145]
Galectin 1+CRP	0.432 (0.275, 0.589) [56]	0.634 (0.457, 0.811) [65]	0.517 (0.408, 0.625) [121]
Galectin 1+CRP (</> = 17.9)	0.488 (0.353, 0.624) [56]	0.623 (0.482, 0.765) [65]	0.541 (0.450, 0.633) [121]

Cut-off with highest correct % classification is 17.9 and 52 for galectin-1 and -3 respectively (for all IBD patients)–hence values above relate to ROC curve for ‘CRP + galectin1 > = 17.9’ versus ‘CRP > 5’ and CRP + galectin3 > = 52’ versus ‘CRP > 5’. The numerical value for the combination of serum CRP and galectin was obtained by simply combining the actual values.

Discussion

In this study, we analysed serum levels of galectins-1, -2, -3, -4, -7 and -8 in IBD patients and healthy controls and investigated their value in determining IBD disease activity. It was found that among these six galectins, galectin-1 and -3 both showed significantly higher serum levels in UC and CD patients than in healthy people.

The intestinal epithelium harbours a number of galectin members and several of them are known to be altered in disease conditions in particularly cancer. Changes of the expression of galectins-1, -3, -4 and -9 in the inflamed tissues of IBD patients were reported previously[26]. Galectin profile analysis at mRNA level with biopsies from patients was able to distinguish IBD from other intestinal inflammatory disorders such as coeliac disease and potentially to also distinguish IBD from active form from quiescent ones[26]. Serum level of galectin-3 was reported in an early study to be higher in IBD patients irrespective of their disease activity[22] but no systematic comparison of the serum levels of galectin members has been done previously in IBD patients. The results in this study shows that levels of galectin-1 and -3, but not galectins-2, -4, -7 and -8, are significantly elevated in the circulation of IBD patients. The increased levels of galectin-1 or -3 however could not separate active and inactive forms of UC and CD. As an exploratory assessment for any indication of increased discrimination, a combination of CRP with galectins1 and 3 was further analysed but neither galectins-1 nor -3 were unable to provide additive sensitivity of CRP in distinguishing active disease from remission. Although further studies with larger sample size are needed to confirm this conclusion, it is possible that the increase in serum galectins might represent an early event in the inflammatory cascade and not subsequently rise further with increase of disease activity. The recent reports showing that several galectin members play key pathogenic roles in animal models of colitis[27] and galectin-3 caused macrophage activation in the induction phase of colitis in a dextran sodium sulphate animal model of colitis[28] are in line with this possibility.

The increased level of serum galectin-3 in IBD patients found in this study is in agreement with an early report showing higher serum galectin-3 level in IBD patients, irrespective of their disease activity, compared with healthy people[22]. It is noted that a recent study by Cibor et al reported significant elevation of serum level of galectin-3-binding protein (Gal-3BP), but not galectin-3 itself, in IBD patients[29]. The much lower median serum galectin-3 level in healthy people (7.1ng/ml) reported in the Cibor study than in this (22.0ng/ml) and a few other studies [30] indicates that bigger cohorts of patients may be needed in future studies to clarify the discrepancy.

It is known that several galectin members including galectin-3[31] and galectin-1 [32, 33] can interact with cell surface glycans and induce endothelium, epithelium or T cell secretion of pro-inflammatory cytokines. For example, galectin-3 interaction with cell surface CD146 (MCAM) on vascular endothelial cells enhances endothelial secretion of IL-6, G-CSF and GM-CSF [34]. Interaction of galectin-1 with intestinal epithelial cells in the presence of TNFα, IL-13 or IL-5 was shown to enhance epithelial secretion of IL-10, IL-25, and TGF-β1[32]. The presence of higher concentrations of galectin-1 caused significant increase of Il-10 secretion from CD4(+) and CD8(+) T-cells[33]. As the chronic inflammatory condition in IBD is closely associated with cytokines[35], an increased secretion of those pro-inflammatory cytokines by circulating galectins-1 and -3, as a result of its elevated level in IBD, may play a role in disease initiation and progression. Moreover, an increased circulation of galectins may themselves have an impact on the pathogenesis and progression of IBD. Indeed, galectin-1 interaction with intestinal epithelial cells was altered in the presence of inflammatory stimuli and modulated cell behaviours [32] and inflammation development[36]. Soluble galectin-3 produced by colon epithelial cells was reported to be a strong activator of colonic lamina propria fibroblasts by inducing cell NF-kappaB activation and IL8 secretion [12].

It should be mentioned that the disease activity used in this study was conventional clinical indices. Patients whose sera were analysed in this study did not undergo endoscopy or faecal collection. As clinical symptoms do not always correlate well with disease activity in IBD and as the clinical indices are less accurate than endoscopy and faecal calprotectin analysis, further investigations to include endoscopy and faecal calprotectin analysis will be needed to determine the possibility of using serum galectins as potential biomarkers in determining IBD disease activity. Further investigations can also include patients with gastrointestinal symptoms such as irritable bowel syndrome and coeliac disease to truly establish the discriminatory value of galectins. Due to the limited number of patients in each sub-group, meaningful evaluation of the impact of disease location (e.g. ileal compared to colonic Crohn’s disease), disease duration and medications on serum galectin values was not possible in this study.

In conclusion, serum levels of galectins-1 and -3, but not galectins-2, -4, -7 and -8, are significantly elevated in both UC and CD patients in comparison to healthy people. Although the increased levels of these galectins are not able to separate active and inactive UC and CD, they have the potential to be developed as biomarkers for general IBD determination.

Galectin-1, -2, -3, -4, -7, -8 expression according to disease duration (S1-S6 Tables), disease location (S7-S12 Tables), medications (S13-18 Tables) and disease severity (S19-S24 Tables).

(DOCX)

Click here for additional data file.

20 Sep 2019

PONE-D-19-23084

SERUM GALECTINS AS POTENTIAL BIOMARKERS OF INFLAMMATORY BOWEL DISEASES

PLOS ONE

Dear Dr Subramanian,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the very constructive points raised during the review process by the two reviewers.

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Reviewer #1: Partly

Reviewer #2: Partly

**********

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Reviewer #2: Yes

**********

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5. Review Comments to the Author

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Reviewer #1: The authors in this study determine the potential for serum galectin-1 and galectin-3 to act as a biomarker for either IBD screening or IBD activity. Previously, tissue levels of galectin-1 and galectin 3 have been shown to be elevated in IBD patients but a systematic comparison of galectin-1 and galectin-3 with other biomarkers has not occurred.

I am concerned by the analysis that uses a CRP threshold of 5mg/dL. The reference that they cite to provide this actually provides data to support a threshold of 5mg/L. This does not negate the findings that serum levels of galectin-1 and -3 are increased in IBD patients but it does affect much of the data analysis.

They do not really describe the issue with these markers being elevated in IBD but not acting as markers of disease activity. Discussion should center around the possibilities that either it is only an early step in the inflammatory cascade or that treatment effect also could cause this to falsely rise (rather than be a marker of inflammation).

Minor points include a few spelling errors and that much of the data provided in the introduction is not appropriately referenced.

Reviewer #2: GENERAL COMMENTS:

The authors presented a relevant work for the field of inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis. They studied whether circulating galectins can be associated with C-reactive protein in diagnosis and/or biomarkers of IBD. In this study, it was evident that serum galectin-1 and -3 levels were increased in serum of patients with IBD in comparison with healthy individuals. On the other hand, the levels of galectin-1, -2, -3, -4, -7 and -8 did not modified in these IBD patients. However, the high levels of galectins were not able of separate active and inactive IBD. The potential to use galectin levels as biomarkers was clearly demonstrated in this manuscript, but the authors need to improve the analysis to try to validate their proposal. Current format is not recommended to publish, although the authors should be encouraged to resubmit after major revisions.

SPECIFIC POINTS PER SECTIONS:

INTRODUCTION

1. The first paragraph needs some references.

2. Some references about galectins and IBD were neglected in the manuscript, such as Sundblad et al., 2018 and Cibor et al., 2019. Please, explore this theme in the introduction section. Moreover, Cibor and colleagues concluded that serum galectin-3 (and Gal-9) should not be considered biomarkers of IBD. Please, make a discussion regarding to galectins as biomarkers of IBD using more recent papers.

MATERIALS AND METHODS

1. Subtitle of the first paragraph is absent

RESULTS

1. Table 1 was appropriately used as base to correlate galectins levels with disease activity and C-reactive protein. However, to amplify the spectrum of analyzed data, the authors should explore underestimated data, such as disease location, disease duration, and medications:

1.1 - Galectin expression and synthesis vary throughout the small and large intestines. Then, disease location can be a relevant factor to be included in the analysis.

1.2 – Disease duration can also be important to study of biomarkers. Galectins have differential expression depending on stage of diseases, such as inflammation and fibrosis.

1.3 – Medications can alter the inflammatory and immunological responses and consequently the expression, synthesis and/or secretion of galectins. Medications can be critical modulators of biomarkers and it is impossible to be neglected in study of biomarkers.

1.4 - Values of serum levels of galectins are missed without a concentration unit as a reference (e.g. ng/mL).

2. Table 2 was excellent to detect serum levels of galectins, in special galectin-1 and -3. However, some information is missing:

2.1 - The insertion of concentration unit (possibly ng/mL) can improve to readers.

2.2 – In table legend, please, include the meaning of “Gal-1 conc”, “Gal-2 conc”, etc.

3. Table 3 was critical to compare active and inactive UC. Again, the insertion of concentration unit (possibly ng/mL) can improve to readers.

4. Table 4 was important to compare active and inactive CD. To better comprehension:

4.1 – Insert the concentration unit (possibly ng/mL) in values of serum galectins;

4.2 – The third column (“All”) shows an asterisk (*). Please, describe the meaning of this symbol.

5. Table 5 showed a comparison with different protocols of analysis using Gal-1 and Gal-3 independently combined with CRP.

5.1 – Describe the unit (possibly ng/mL);

5.2 – It was not clear the strategy to obtain the values (“mean” and “median”). Please, describe in the text or in materials and methods section

6. Table 6 showed a ROC analysis. However, the text subsequent (below of table) can be reorganized to explain the values (unit) and symbols (“>=” is equivalent to “≥”?).

DISCUSSION

1. Selection criteria for using galectin-1, -2, -3, -4, -7 and -8 should be discussed here. Why other galectins were not investigated in IBD patients? At least, these arguments can be detailed in materials and methods section.

2. The authors discussed about immunomodulatory roles of galectin-3 inducing pro-inflammatory signals (such as IL-6 dependent manner). These aspects can be explored with more details, including galectin-1 in the context. Several cytokines are associated with galectin-1 and galectin-3 levels in IBD. Imbalances between pro and anti-inflammatory responses seem relevant to insert both galectins as biomarkers.

3. There are several neglected works using galectins to study the pathogenesis and progression of IBD. The insertion in discussion section will improve the quality of this manuscript.

**********

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Reviewer #1: No

Reviewer #2: No

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22 Oct 2019

Dear Dr. Chammas,

Thanks for sending us the reviewers’ very helpful comments. We have made substantial revision of the manuscript (including the addition of 18 new tables as supplementary supporting materials and also addition of 11 new references) according to the referees’ suggestions and comments including the addition of 18 new tables in supplement materials. Taking the reviewers’ points in turns:

Journal requirements:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

We have carefully edited the manuscript in accordance to the style requirements of PLOS One

2. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified (1) whether consent was informed and (2) what type you obtained (for instance, written or verbal). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information.

Written informed consent was obtained from all participants in this study and the study population was comprised of adults over the age of 18. We have included these statements in the Method section (page 7).

3. Thank you for stating the following in the Competing Interests section:

"TBY, SD, NM and JR declare no conflicts of interest.

SS has received speaker fee from MSD, Actavis, Abbvie, Dr Falk pharmaceuticals, Shire and received educational grant from MSD, Abbvie.

LGY is an Academic Editor of PLOS ONE"

Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

This has been revised according to the suggestions (p19)

4. Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf.

Updated Competing Interest statement is included: TBY, SD, NM and JR declare no conflicts of interest. SS has received speaker fee from MSD, Actavis, Abbvie, Dr Falk pharmaceuticals, Shire and received educational grant from MSD, Abbvie. LGY is an Academic Editor of PLOS ONE. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Reviewer 1 comments:

I am concerned by the analysis that uses a CRP threshold of 5mg/dL. The reference that they cite to provide this actually provides data to support a threshold of 5mg/L. This does not negate the findings that serum levels of galectin-1 and -3 are increased in IBD patients but it does affect much of the data analysis.

They do not really describe the issue with these markers being elevated in IBD but not acting as markers of disease activity. Discussion should center around the possibilities that either it is only an early step in the inflammatory cascade or that treatment effect also could cause this to falsely rise (rather than be a marker of inflammation).

Thanks the reviewer for spotting the Unit typo which has been corrected in the revision (5mg/L)

As suggested, we have added the below paragraph to discuss the discovery that galectins are elevated in IBD but are not effective markers of disease activity. “Although further studies with larger sample size are needed to confirm this conclusion, it is possible that the increase in serum galectins might represent an early event in the inflammatory cascade and not subsequently rise further with increase of disease activity. The recent reports showing that several galectin members play key pathogenic roles in animal models of colitis [new ref 27] and galectin-3 caused macrophage activation in the induction phase of colitis in a dextran sodium sulphate animal model of colitis[new ref 28] are in line with this possibility” (p17).

Minor points include a few spelling errors and that much of the data provided in the introduction is not appropriately referenced.

All the spelling errors in the manuscript have been corrected and three new references (refs 1 to 3) have been added in the introduction (p4)

Reviewer #2: GENERAL COMMENTS:

INTRODUCTION

1. The first paragraph needs some references.

Several new references were added (refs 1-4, p4)

2. Some references about galectins and IBD were neglected in the manuscript, such as Sundblad et al., 2018 and Cibor et al., 2019. Please, explore this theme in the introduction section. Moreover, Cibor and colleagues concluded that serum galectin-3 (and Gal-9) should not be considered biomarkers of IBD. Please, make a discussion regarding to galectins as biomarkers of IBD using more recent papers.

These suggested papers have all been discussed in the study and references included

MATERIALS AND METHODS

1. Subtitle of the first paragraph is absent

A subtitle of “Materials” has been added in the revision (p7)

RESULTS

1. Table 1 was appropriately used as base to correlate galectins levels with disease activity and C-reactive protein. However, to amplify the spectrum of analyzed data, the authors should explore underestimated data, such as disease location, disease duration, and medications:

We have conducted substantial additional analysis of serum galectin levels in accordance to disease location, disease duration and medications and included the results as 18 supplemental tables (Tables S1 to S18). These analyses showed no significant differences in median serum galectin values of any of the galectin members- 1, -2, -3, -4, -7 and -8 when stratified by disease location, duration or medications. We have included the results of those new analysis as supplement materials (Table S1 to S18) and add a statement in the main text as “We also analysed the serum levels of galectins-1, -2, -3, -4, -7, and -8 according to disease location (Table S1-S6), disease duration (Table S7-S12) and concomitant medications (Table S13-S18). No significant differences in median galectin values across the variables was observed for any galectin members (Table S1 to S18). As the sample size in each sub-group in this study was small, future studies with larger sample numbers would help to clarify this conclusion” (p11)

1.1 - Galectin expression and synthesis vary throughout the small and large intestines. Then, disease location can be a relevant factor to be included in the analysis.

1.2 – Disease duration can also be important to study of biomarkers. Galectins have differential expression depending on stage of diseases, such as inflammation and fibrosis.

1.3 – Medications can alter the inflammatory and immunological responses and consequently the expression, synthesis and/or secretion of galectins. Medications can be critical modulators of biomarkers and it is impossible to be neglected in study of biomarkers.

All have been revised as discussed above

1.4 - Values of serum levels of galectins are missed without a concentration unit as a reference (e.g. ng/mL).

Values of the serum galectin levels was added (ng/ml, Tables 2-4)

2. Table 2 was excellent to detect serum levels of galectins, in special galectin-1 and -3. However, some information is missing:

2.1 - The insertion of concentration unit (possibly ng/mL) can improve to readers.

2.2 – In table legend, please, include the meaning of “Gal-1 conc”, “Gal-2 conc”, etc.

3. Table 3 was critical to compare active and inactive UC. Again, the insertion of concentration unit (possibly ng/mL) can improve to readers.

Galectin concentration unit (ng/ml) was added and the word “conc” was removed to make it clearer

4. Table 4 was important to compare active and inactive CD. To better comprehension:

4.1 – Insert the concentration unit (possibly ng/mL) in values of serum galectins;

4.2 – The third column (“All”) shows an asterisk (*). Please, describe the meaning of this symbol.

Concentration unit (ng/ml) was included and a footnote was added for the asterisk “One patient whose disease activity could not be accurately established was also included in this analysis”

5. Table 5 showed a comparison with different protocols of analysis using Gal-1 and Gal-3 independently combined with CRP.

5.1 – Describe the unit (possibly ng/mL);

Concentration unit (ng/ml) was added

5.2 – It was not clear the strategy to obtain the values (“mean” and “median”). Please, describe in the text or in materials and methods section

This has now been clarified in the methods section with the addition of the following sentence: Mean and median serum galectin levels were calculated to account for inter-individual variability in serum galectin levels (p8).

5. Table 6 showed a ROC analysis. However, the text subsequent (below of table) can be reorganized to explain the values (unit) and symbols (“>=” is equivalent to “≥”?).

We have now clarified this by the addition of the following to the footnote of the table: The numerical value for the combination of serum CRP and galectin was obtained by simply combining the actual values.

DISCUSSION

1. Selection criteria for using galectin-1, -2, -3, -4, -7 and -8 should be discussed here. Why other galectins were not investigated in IBD patients? At least, these arguments can be detailed in materials and methods section.

The selection of these galectin members was entirely due to the commercial availability of the detection agents (capture and detection anti-galectin antibodies and recombinant galectins) of the sandwich galectin ELISA. We have made this clearer in the Method section (p8)

2. The authors discussed about immunomodulatory roles of galectin-3 inducing pro-inflammatory signals (such as IL-6 dependent manner). These aspects can be explored with more details, including galectin-1 in the context. Several cytokines are associated with galectin-1 and galectin-3 levels in IBD. Imbalances between pro and anti-inflammatory responses seem relevant to insert both galectins as biomarkers.

As suggested, we have added three new paragraphs in the Discussion section to discuss the immunomodulatory roles of galectin-1 and -3 and the galectin-associated cytokines in IBD (p17-19) and included a few new references (refs 27-30).

3. There are several neglected works using galectins to study the pathogenesis and progression of IBD. The insertion in discussion section will improve the quality of this manuscript.

As suggested, we have discussed the few early studies about the role of galectin-1 and -3 in IBD pathogenesis and progression in the discussion section and included a few new references (ref 27 to 31)

We hope the addition of the new data and the substantial revision has satisfactorily addressed the reviewers’ comments and the manuscript is now in an acceptable form for publication by PLOS One.

Yours sincerely,

Sreedhar Subramanian

Submitted filename: Sree-IBD MS-response to reviewers comments.docx

Click here for additional data file.

5 Nov 2019

PONE-D-19-23084R1

SERUM GALECTINS AS POTENTIAL BIOMARKERS OF INFLAMMATORY BOWEL DISEASES

PLOS ONE

Dear Dr Subramanian,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Dec 20 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Roger Chammas, M.D, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Reviewer 1 still has some concerns that need to be addressed before the ms is considered acceptable. Please, address the constructive issues raised and submit a revised version of your ms.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In the materials and methods section, there is a statement that "CRP>=5mg/L was used to define active disease" although I believe the clinical indices were actually used to define activity or inactivity.

I'm a bit concerned about the methods to add CRP and galectin-1 or galectin-3 to create a composite score to analyze against active or inactive IBD as it might underestimate the potential finding regarding an improvement in sensitivity for active IBD if you simply use the dichotomous variable of "galectin-1 normal or abnormal" and similarly for galectin-3.

With regard to the interesting finding that neither galectin-1 or galectin-3 associate with disease activity, it's not clear what the spectrum of disease activity (based on HBI and SCCAI) was. If there is a predominance of mildly active patients, that may explain this finding as patients with moderate-severe activity might be more likely to have higher serum biomarkers than patients in the inactive state.

Reviewer #2: The authors followed all suggestions. Current form is extremely relevant to review possible correlation between galectins and inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis. This version contemplates an extensive analysis of data regarding to circulating galectins and biomarkers of IBD. Discussion about increase of galectin-1 and -3 serum levels in patients with IBD was substantially improved in comparison with first version of the review. The potential to use galectin levels as biomarkers was clearly demonstrated in this manuscript. Given that authors satisfactorily responded the reviewer's comments, added the relevance of the theme, current format can be recommended to publication.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

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26 Nov 2019

Dear Dr. Chammas,

Thanks for giving us the opportunity to address the reviewers’ further comments to this manuscript. We have revised the manuscript in accordance to these comments and included six additional tables in the supplement materials. Take these comments in turns:

Reviewer 2:

In the materials and methods section, there is a statement that "CRP>=5mg/L was used to define active disease" although I believe the clinical indices were actually used to define activity or inactivity.

Clinical indices were indeed used to define disease activity. We however also used a CRP threshold of >=5mg/L to compare the diagnostic sensitivity of CRP compared to serum galectins in differentiation of active from inactive disease. To make this clearer, we have added below in the revision: ‘We investigated the ability of CRP to distinguish active disease from remission using a threshold of CRP≥5mg/L, as described previously’ in the method section (page 7). (please also see responses below).

I'm a bit concerned about the methods to add CRP and galectin-1 or galectin-3 to create a composite score to analyze against active or inactive IBD as it might underestimate the potential finding regarding an improvement in sensitivity for active IBD if you simply use the dichotomous variable of "galectin-1 normal or abnormal" and similarly for galectin-3.

We acknowledge the limitations of combining serum CRP to galectins-1 and -3 to produce a composite measure hence included the below in the discussion section to make it clearer the exploratory nature of this combination of analysis: “As an exploratory assessment for any indication of increased discrimination, a combination of CRP with galectins1 and 3 was further analysed but neither galectins-1 nor -3 were unable to provide additive sensitivity of CRP in distinguishing active disease from remission” (p17).

With regard to the interesting finding that neither galectin-1 or galectin-3 associate with disease activity, it's not clear what the spectrum of disease activity (based on HBI and SCCAI) was. If there is a predominance of mildly active patients, that may explain this finding as patients with moderate-severe activity might be more likely to have higher serum biomarkers than patients in the inactive state.

To address this comment, we have now performed additional analyses of serum galectins-1 and -3 across the spectrum of disease activity. We used previously defined thresholds of Harvey-Bradshaw index and simple clinical colitis activity index to define clinical severity. Both in CD and UC, there were approximately equal number of patients with mild and moderate disease activity respectively. Only one patient had severe disease. We have included the results of these analysis in six new tables in the supplemental materials (Tables S19-S24) and added below in in the results section: ‘We also analysed serum galectin levels according to the clinical severity of disease activity (remission, mild, moderate or severe). No association was observed between serum galectin levels and increasing disease severity” (p12).

We hope these revisions have satisfactorily addressed all the reviewers’ comments and the manuscript is now in an acceptable form for publication.

With many thanks,

Sreedhar Subramanian

On behalf of all co-authors

Submitted filename: Sree-IBD-revision-2-Response to reviewers conments.docx

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9 Dec 2019

PONE-D-19-23084R2

SERUM GALECTINS AS POTENTIAL BIOMARKERS OF INFLAMMATORY BOWEL DISEASES

PLOS ONE

Dear Dr Subramanian,

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Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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Reviewer #2: Yes

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Reviewer #1: The authors have responded to all of my concerns in a satisfactory manner. I have no additional concerns.

Reviewer #2: This current version is appropriate to be published. The second revision improved the quality of manuscript and contemplated an extensive analysis of data regarding to circulating galectins and biomarkers of IBD. Possible correlation between galectins and inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis, is extremely relevant. Moreover, the potential to use galectin levels as biomarkers was clearly demonstrated in this manuscript. Given that authors satisfactorily responded the reviewer's comments, current format is recommended to publication.

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Reviewer #1: No

Reviewer #2: Yes: Felipe Leite de Oliveira

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Submitted filename: Reviewer Recommendation and Comments for Manuscript Number PONE-D-19-23084R2.pdf

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9 Dec 2019

Dear Dr. Roger Chammas,

Thanks for sending us the reviewers’ comments on the second round revision of this manuscript. We are pleased to see that both reviewers are happy with the second revision and have recommended publication of the manuscript, see below:

Reviewer #1: The authors have responded to all of my concerns in a satisfactory manner. I have no additional concerns.

Reviewer #2: This current version is appropriate to be published. The second revision improved the quality of manuscript and contemplated an extensive analysis of data regarding to circulating galectins and biomarkers of IBD. Possible correlation between galectins and inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis, is extremely relevant. Moreover, the potential to use galectin levels as biomarkers was clearly demonstrated in this manuscript. Given that authors satisfactorily responded the reviewer's comments, current format is recommended to publication.

The point about CRP and galectin-1/-3 mentioned in your decision letter was raised by Reviewer 1 in the first round review which had been adequately addressed in the second revision as the reviewer clearly stated in their comments. Both reviewers also made it clear that the second revision is satisfactory and no further revision is required.

It looks that the reviewers’ comments in the first review round somehow corrupted into the system of the second review round which created the confusion for the request of a point already satisfactorily addressed in the new decision letter.

We hope this letter helps to clarify the situation and the manuscript is ready to be accepted for publication in PLOS One.

Yours sincerely,

Sreedhar Subramanian, MD, MRCP

On behalf of all co-authors

Submitted filename: Sree-IBD-revision-2-Response to reviewers conments.docx

Click here for additional data file.

17 Dec 2019

SERUM GALECTINS AS POTENTIAL BIOMARKERS OF INFLAMMATORY BOWEL DISEASES

PONE-D-19-23084R3

Dear Dr. Subramanian,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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With kind regards,

Roger Chammas, M.D, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

31 Dec 2019

PONE-D-19-23084R3

Serum galectins as potential biomarkers of inflammatory bowel diseases

Dear Dr. Subramanian:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Academic Editor

PLOS ONE