The expansion of CD14+ CD163+ subpopulation of monocytes and myeloid cells‐associated cytokine imbalance; candidate diagnostic biomarkers for celiac disease (CD)

Abstract

Celiac disease (CD) is a chronic autoimmune disorder of small intestine against dietary gluten, among genetically predisposed individuals. Monocytes are versatile innate immune cells involved in the regulation of inflammation, and strongly involved in the intestinal immunity. However, the role of monocytes and their subtypes in CD is not well demonstrated.

Methods

Here, we assessed the polarization of CD14+ monocytes by evaluating the M1 (CD16) and M2 (CD163) markers by flowcytometry, their soluble forms (sCD16 and sCD163), and the serum levels of IL‐10, IL‐12, TGF‐β, and TNF‐α cytokines using ELISA method, among 30 CD patients and 30 sex‐ and age‐matched healthy subjects (HS). We also analyzed the diagnostic values of all variables with significant differences.

Results

CD14+CD163+ monocytes were more frequent in CD patients than HS, while CD14+CD16+ monocytes were higher in HS. IL‐10and TNF‐α increased, and TGF‐β expression was decreased among CD patients. The sCD16 serum levels were elevated in patients, while sCD163 was higher but not significant among CD patients. CD163+/CD16+ and IL‐10/IL‐12 ratios were higher in CD patients, and TGFβ/TNFα ratio was higher in HS group. IL‐10, CD14+CD163+, TNF‐α, and IL‐10/IL‐12 ratios with the AUC over 0.7 were introduced as fair diagnostic markers. Our findings revealed that the M2 (CD14+CD163+) monocytes were more frequent among CD patients, and the cytokine balance was disturbed.

Conclusion

According to the significant functional diversities of monocyte subtypes between CD patients and HS group, these immunologic markers could be introduced as specific diagnostic biomarkers for CD.

CD14+CD163+ monocytes were more frequent in CD patients than HS, while CD14+CD16+ monocytes were higher in HS. IL‐10, CD14+CD163+, TNF‐α, and IL‐10/IL‐12 ratios with the AUC over 0.7 were introduced as fair diagnostic markers. Our findings revealed that the M2 (CD14+CD163+) monocytes were more frequent among CD patients and the cytokine balance was disturbed. According to the significant functional diversities of monocyte subtypes between CD patients and HS group, these immunologic markers could be introduced as specific diagnostic biomarkers for CD.

1. INTRODUCTION

Celiac disease (CD) is a common, chronic, and permanent autoimmune disorder of small intestine against dietary gluten of wheat, rye, and barley. ¹ CD is a complex inflammatory condition in which the main genetic predisposing factors (HLA‐DQ2 and HLA‐DQ8), the associated auto‐antigen (tissue transglutaminase(tTG)), and the environmental trigger (gluten) are all introduced. ² CD could also increase the risk of other autoimmune diseases such as type 1 diabetes ³ and Graves’ disease. ⁴ The prevalence of CD is approximately 0.5–1% worldwide and is rapidly rising due to the uncontrolled spread of diets comprising of high gluten quantities. ⁵

Despite the expansion of clinical notice and the development of rather sensitive serological screening tests, CD is associated with a wide range of unspecific clinical symptoms including diarrhea, abdominal pain, and severe malnutrition, which may lead to challenges during diagnosis. ⁶ Moreover, a large number of patients develop non‐digestive symptoms such as anemia, osteoporosis, or arthritis, ⁷ Accordingly, the disease may remain undiagnosed, especially during the first stages. ⁸ Moreover, CD should be confirmed with intestinal biopsies, which is an invasive and also prohibitive method. ⁹ Therefore, more specific tests are needed to diagnose CD in the first stages and also confirm after screening the patients, and immunopathogenesis‐specific markers could be reliable tools in this manner.

The immunopathogenesis of CD is well defined and includes the intensified CD4+ T cell and highly disease‐specific B‐cell responses against modified gluten and the self‐protein of transglutaminase 2 (TG2). ¹⁰ Moreover, the HLA‐DQ molecules are responsible in the pathogenesis of CD by specific delivery of gluten antigens to CD4+ T cells. ¹¹ However, the role of innate immunity in CD is underestimated, while innate immune cells could not only regulate adaptive immune response, but also function against the persistent inflammatory condition in CD. ¹²

Monocytes and macrophages are innate immune phagocytic cells involved in the regulation of immune response during inflammation, tissue repair, and pathogenesis of several autoimmune diseases. ¹³ , ¹⁴ Monocytes, which may differentiate into macrophages after infiltration into the lamina propria, are strongly involved in the intestinal immunity by secretion of cytokines or direct interaction with intestinal epithelial cells (IEs). ¹⁵ It has been reported that gluten‐exposed monocytes, isolated from CD patients, produced elevated levels of pro‐inflammatory cytokines including TNF‐α and IL‐8. ¹⁶ Moreover, IL‐15 as a celiac‐simulating cytokine was capable of stimulating normal monocytes in secreting pro‐inflammatory cytokines. ¹⁷

Activated monocytes and macrophages are plastic cells, which may polarize into classically activated (M1) cells during infection with giving raise to the pro‐inflammatory cytokines and, alternatively activated (M2) cells associated with tissue repair and resolving inflammation. ¹⁸ According to the significant immunophenotypic and functional differences between these two subtypes of monocytes, they play different roles in GUT‐associated inflammatory disorders such as asmalignancies, ¹⁹ irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), ²⁰ and autoimmune conditions including CD. ²¹ The immunophenotyping of monocytes in CD patients and characterizing their functions by assessing the secretion of major pro‐ and anti‐inflammatory cytokines could better clarify the role of monocytes in CD pathogenesis and might introduce better diagnostic utilities. Several M1‐ and M2‐associated markers have been introduced to date, of which CD163 (M2 marker), a scavenger receptor participating in the removal of dying cells, ²² and CD16 (FcγRIII; M1 marker) ²³ as a glycoprotein expressed on the surface of monocytes involved in stimulating inflammatory immune responses, are more tightly associated with the abovementioned subtypes. Therefore, we aimed to assess the polarization of CD14+ monocytes by evaluating the expression of M1 (CD16) and M2 (CD163) markers, the secretion levels of the soluble forms of these markers (sCD16 and sCD163), and the serum levels of IL‐10, IL‐12, TGF‐β, and TNF‐α cytokines in CD patients and healthy subjects. Finally, we aimed to analyze the diagnostic values of all variables with significant differences between two groups of CD patients and healthy subjects.

2. MATERIALS AND METHODS

2.1. Patients and controls

A total of 30 celiac disease patients (22 female and 8 male), according to the clinical examinations, history of the patients, and laboratory measures consisting of positive anti‐transglutaminase antibodies and endomysial antibody, confirmed by an expert specialist and 30 age‐ and sex‐matched healthy volunteers were recruited from the department of gastroenterology, Rohani hospital, Babol University of Medical Sciences, Babol, Iran. The mean age of CD patients was 35.57 ± 10.92, while it was 36.03 ± 11.18 for the control group. All participants diagnosed with or claimed to suffer from primary or secondary immunodeficiency, inflammatory, or infectious diseases (at the time of enrollment in the research project), malignancies, or any other autoimmune diseases were not included. Moreover, no pregnant female participant was included in the study, and blood samples were taken during the first week of follicular phase of menstrual cycle (between day 0 and 7) from female individuals. The patients were all on a gluten‐free diet for at least 6 months prior to the study. In order to better represent the clinical status of participants regarding the CD‐associated variables and markers, all available laboratory data of CD patients and normal subjects are listed in Table 1, and significant differences between each two groups are calculated. The current study was authorized by the committee of ethics at Golestan University of Medical Sciences, and an informed consent was signed by all participators after careful explanation of the research, according to the regulations stated within the Declaration of Helsinki. ²⁴ Roughly, 5 ml of peripheral blood was collected from all participators and immediately aliquoted into two sterile tubes; one of which (2 ml) was added to an anti‐coagulated tube used for further immunophenotyping examinations, and the second one (3 ml) was used for serum isolation and subsequent ELISA measurements. The samples were then transferred to Amirkola Shafizadeh Pediatric Hospital, Babol University of Medical Sciences for flowcytometric immunophenotyping analyses of monocytes, while separated sera were preserved at −80℃ until use.

TABLE 1.

Laboratory findings of celiac disease patients and healthy subjects

Characteristics*	CD patients (N = 30)	Healthy subjects (N = 30)	p‐value
WBC (109/L)	8.42 ± 3.31	7.12 ± 1.70	0.016
RBC (106/ml)	4.59 ± 0.46	5.11 ± 0.75	0.048
Hb (g/dl)	12.12 ± 1.39	13.08 ± 1.65	0.220
HCT (%)	36.77 ± 3.65	39.66 ± 4.70	0.108
MCV (g/dl)	80.47 ± 8.72	83.70 ± 3.66	0.024
MCH (pg)	26.55 ± 3.38	27.90 ± 1.51	0.046
RDW (%)	13.12 ± 1.48	12.91 ± 0.69	0.003
anti‐tTG, IgA (U/ml)	76.64 ± 66.10	0.48 ± 0.24	≤0.001
ALP (IU/L)	445.04 ± 161.28	292.11 ± 162.98	0.775
25‐hydroxy vitamin D (nMol/L)	31.74 ± 14.99	28.70 ± 9.90	0.012
Amylase (U/L)	66.00 ± 32.97	72.00 ± 42.04	0.833
Lipase (U/L)	16.50 ± 0.71	13.67 ± 7.37	0.097
Folate (Folic Acid) (ng/ml)	8.00 ± 4.58	15.40 ± 5.57	0.681
Vit B12 (pg/ml)	215.00 ± 63.84	369.33 ± 55.87	0.793
Ferritin (µg/L)	26.73 ± 15.39	81.25 ± 41.03	0.040
Mg (mmol/L)	2.20 ± 0.14	2.00 ± 0.10	0.576
Zn (µg/dl)	75.50 ± 13.43	105.00 ± 7.07	0.684
EMA titer
Negative	0 (0%)	30 (100%)	‐
1/30	23 (76.7%)	0 (0%)	‐
1/100	4 (13.3%)	0 (0%)	‐
1/300	3 (10%)	0 (0%)	‐

ALP, alkaline phosphatase; Anti‐TPO, antithyroid peroxidase antibody; anti‐tTG IgA, tissue transglutaminase (tTG) antibody, IgA; EMA, endomysial (EMA) IgG antibody. Hb, hemoglobin; HCT, hematocrit; MCH, mean corpuscular hemoglobin; MCV, mean corpuscular volume; Mg, magnesium; RBC, red blood cells; RDW, red blood cell distribution width; Vit B12, vitamin B12; WBC, white blood cells; Zn, Zinc.

Significant differences are shown in bold and italics.

^*Data were demonstrated as Means ± STDEV (standard deviation) or number (percentage).

2.2. Immunophenotyping of monocytes by flowcytometry

The cell surface staining of freshly prepared whole blood samples was conducted using PE‐conjugated anti‐human CD14 antibody (Cat # 301806; Biolegend), FITC‐conjugated anti‐human CD16 antibody (Cat # 302006; Biolegend), and PerCP‐conjugated anti‐human CD163 antibody (Cat # 333626; Biolegend) markers, as previously described. ¹⁴ Partec PAS III flow cytometer (Partec) and FlowJo 7.6.1 software (FlowJo LLC) were used to evaluate the immunophenotypes of all stained samples and examining the expansion of monocytes subpopulations in celiac disease patients and healthy subjects. As demonstrated in Figure 1A‐F, PE‐CD14 antibody was used to gate (R1), separated monocytes (Figure 1A and D) in FL2(Figure 1B and E). FITC‐CD16 and PerCP‐CD163 antibodies were used to stain CD14+ monocytes and detected in FL1 and FL3 (Figure 1C and F). Q4 quadrant demonstrated CD14+CD163+ monocytes, while Q2 showed CD14+CD16+ cells.

FIGURE 1.

Immunophenotyping of CD14+ CD16+ monocytes and CD14+ CD163+ monocytes in celiac disease patients in comparison with the healthy controls. PE‐conjugated anti‐human CD14 antibody was used to gate (R1), separated monocytes (A, D) in FL2 (B, E). FITC‐conjugated anti‐human CD16 antibody and PerCP‐conjugated anti‐human CD163 antibody were used to stain CD14+ monocytes and detected in FL1 and FL3 (C, F). Q4 shows CD14+CD163+ monocytes, while Q2 demonstrates CD14+CD16+ cells. Mann‐Whitney U results showed that CD163+ monocytes were significantly more frequent in celiac disease patients (CDP) than healthy subjects (HS). Although not statistically significant, the population of CD16+ monocytes were more frequent in HS group. Each bar represents Means ±Standard deviation

2.3. ELISA assays

The commercially available ELISA kits were used to assess the serum levels of sCD163 and sCD16 among celiac disease patients and healthy subjects (Bioassay Technology Laboratory). Moreover, the serum levels of selected pro‐ and anti‐inflammatory cytokines (IL‐10, IL‐12, TGF‐β1, and TNF‐ α) were evaluated by commercially available ELISA kits (Biolegend), following the manufacturer's protocols. We also considered various aspects affecting the efficacy of ELISA assay. ²⁵ All experiments were conducted in triplicates, and the results were shown as nanograms per ml (ng/ml). Finally, we applied linear regression analysis ²⁶ to quantify the absorbance of samples at 450 nm against the reference absorbance of 570 nm using a Stat Fax 4200 microplate reader (Awareness Technology).

2.4. Statistical analyses

SPSS 23.0 and GraphPad Prism 8.4.2 statistical software were used to analyze the acquired data and prepare graphs. The Independent Samples t test or Mann‐Whitney U (its nonparametric alternative) tests were employed to compare the differences between experimental counterparts. Receiver operator characteristic (ROC) curve analyses were performed to evaluate the diagnostic value of significant variables, ²⁷ distinguishing celiac disease patients from healthy subjects. p‐values lower than 0.05 were considered as statistically significant.

3. RESULTS

3.1. The expansion of CD14+CD163+ subpopulation of monocytes in celiac disease patients

We evaluated the percentages of CD16+ and CD163+ cells among CD14+ monocytes in peripheral blood of celiac disease patients in comparison with the healthy subjects. As shown in Figure 1, CD14+CD163+ monocytes were significantly more frequent in celiac disease patients than healthy subjects (p‐value = 0.001) (Figure 1G). Although not statistically significant, the population of CD14+CD16+ monocytes were more frequent in healthy subjects.

3.2. The altered serum levels of pro‐ and anti‐inflammatory cytokines

ELISA cytokine assay was performed to assess the levels of IL‐10, IL‐12, TGF‐β, and TNF‐α in the sera of CDPs in comparison with the HS. As demonstrated in Figure 2, IL‐10 was significantly increased among patients (p‐value = 0.001) (Figure 2A). IL‐12 was also elevated in the sera of CDPs, which was not statically significant (Figure 2B). TGF‐β expression was markedly decreased in CDPs (p‐value = 0.011) (Figure 2C), while TNF‐α expression was noticeably elevated among patients (p‐value = 0.002) (Figure 2D).

FIGURE 2.

Cytokine analyses of IL‐10 (A), IL‐12 (B), TGFβ (C), and TNFα (D) in the sera of celiac disease (CD) patients and healthy controls (HS). Independent Samples t test or Mann‐Whitney U results revealed that IL‐10 and TNFα were significantly higher in the sera of CD patients while TGFβ was expressed lower in this group. Each bar represents Means (the numbers annotated within each bar) ± Standard deviation (error bars)

3.3. The elevation of soluble CD16 (sCD16) among patients

ELISA assay was also conducted to evaluate the secretion of sCD16 and sCD163 in the sera of CDPs and healthy subjects. As represented in Figure 3, sCD16 was significantly elevated in CDPs (p‐value = 0.001) (Figure 3A), while sCD163 was higher but not significant among patients (Figure 3B). We also performed a spearman correlation study between the percentages of CD14+CD16+ population of monocytes vs sCD16 serum levels and the percentages of CD14+CD163+ population of monocytes vs sCD163 serum concentrations, which did not result in significant associations (data not shown).

FIGURE 3.

Secretion level of sCD16 (A) and sCD163 (B) in the sera of celiac disease (CD) patients and healthy controls (HS). Mann‐Whitney U results showed that sCD16 (A) was significantly higher in CD patients, while sCD163 (B) was higher but not significantly among patients. Each bar represents Means (the numbers annotated within each bar) ± Standard deviation (error bars). p‐values smaller than 0.05 are considered as significant

3.4. Characterization of M2/M1 balance in peripheral blood monocytes

In order to characterize the M2/M1 balance in peripheral blood monocytes, CD163+/CD16+, IL‐10/IL‐12, TGFβ/TNFα, and sCD16/sCD163 ratios were defined and compared between CDPs and HS. Our findings revealed that CD163+/CD16+ (p‐value = 0.016) (Figure 4A) and IL‐10/IL‐12 (p‐value = 0.003) (Figure 4B) ratios were significantly higher in CDPs, while TGFβ/TNFα ratio (p‐value = 0.010) (Figure 4C) was higher in HS group. Although sCD16/sCD163 ratio (Figure 4D) was also higher in healthy subjects, the difference was not statistically remarkable (p‐value = 0.626).

FIGURE 4.

Characterization of M2/M1 balance in peripheral blood monocytes by defining the CD163+/CD16+ (A), IL‐10/IL‐12 (B), TGFβ/TNFα (C), and sCD16/sCD163 (D) ratios between celiac disease (CD) patients and healthy controls (HS). Mann‐Whitney U results revealed that CD163+/CD16+ (A) and IL‐10/IL‐12 (B) ratios were significantly higher in CD patients while TGFβ/TNFα ratio (C)was bigger in HS group. Each bar represents Means (the numbers annotated within each bar) ± Standard deviation (error bars). p‐values smaller than 0.05 are considered as significant

3.5. The diagnostic value of potential biomarkers in celiac diseases

We assessed the diagnostic values of all variables with significant differences between two groups of CDPs and healthy subjects. Accordingly, ROC curve analyses were conducted, area under the curves (AUCs), cutoff values, specificity, sensitivity, and likelihood ratios were calculated and reported for each candidate biomarker. Figure 5 demonstrates all fair biomarkers with the AUC ≥0.7. As illustrated in Figure 5A, the serum level of IL‐10 was considered to be a potential biomarker in distinguishing CD patients from healthy subjects. AUC for IL‐10 serum level was 0.7595 (95% CI, 0.6301 to 0.8889; p = 0.0007). Setting the optimal cutoff point at 23.40 exhibited a sensitivity of 78.57% and a specificity of 70.00% with the likelihood ratio (LR) of 2.619. AUC for CD14+CD163+ population of monocytes was 0.7478 (95% CI, 0.6168 to 0.8787; p = 0.001) (Figure 5B). Setting the favored cutoff point at 86.50 gave a sensitivity of 76.67% and a specificity of 66.67% with the LR of 2.30. Similarly, the AUC for TNF‐α serum level, as an acceptable biomarker for CD, was 0.7268 (95% CI, 0.5926 to 0.8610; p = 0.003) (Figure 5C). Setting the optimum cutoff point at 16.13 resulted in a sensitivity of 86.67% and a specificity of 60.71%, with the LR of 2.21. Similarly, the AUC for IL‐10/IL‐12 ratio was 0.6976 (95% CI, 0.5580 to 0.8373; p = 0.0098) (Figure 5D). Setting the favorable cutoff point at 3.028 demonstrated a sensitivity of 63.33% and a specificity of 82.14% with the LR of 3.55.

FIGURE 5.

ROC curve analysis of possible diagnostic markers. Area under the curve (AUC), p‐value, cutoff value, specificity, sensitivity, and likelihood ratios were calculated and reported for each variable. p‐values smaller than 0.05 are considered as significant. The potential biomarkers with AUCs equal to or bigger than 0.7 are listed as good biomarkers and illustrated. Celiac disease patients, CDP, healthy subjects, HS

4. DISCUSSION

Celiac disease (CD) is a gluten‐induced autoimmune enteropathy among genetically predisposed individuals with complex clinical manifestations, making the specific diagnosis difficult, especially at early and asymptomatic stages. ²⁸ The immunopathogenesis of CD, which could be employed as an efficient diagnostic tool, is thoroughly investigated regarding the adaptive immunity and the role of T cells, NKT cells, and B lymphocytes of the intestinal mucosa in mediating the inflammation has been highlighted. ²⁹ However, the role of innate immunity in this regard is not well described and needs careful reconsiderations. Monocytes, as major innate immune cells, could play a significant role in the immunopathogenesis of CD. ¹³ These versatile precursors of phagocytes are called to the inflammatory milieu by responding to certain cytokines and chemokines, and form macrophages and dendritic cells in the inflamed tissue. ³⁰ The macrophages could be activated and polarized into different M1 (pro‐inflammatory) and M2 (anti‐inflammatory) subtypes by secreting relevant cytokines. ³⁰ It has been reported that gliadin peptides are capable of activating blood monocytes from CD patients, which determines the significance of these cells in CD immunopathogenesis. ¹⁶ Moreover, Zanoni et al. ³¹ revealed that a subset of autoantibodies against transglutaminase could bind to TLR4 (Toll‐Like Receptor 4) and induce the activation of monocytes in CD. According to the important role of monocytes and macrophages in controlling intestinal inflammation, these aberrations have been also observed in similar disorders such as IBD and IBS. ²⁰ Furthermore, IL‐15, which is a major contributor in the immunopathogenesis of CD, could differentiate monocytes and determine Th17 and Th1 responses to wheat gliadin. ¹⁷ In the present study, we evaluated the polarization capacity of CD14+ monocytes by evaluating the expression of M1 (CD16) and M2 (CD163) markers, the secretion levels of the soluble forms of these markers (sCD16 and sCD163), and the serum levels of IL‐10, IL‐12, TGF‐β, and TNF‐α cytokines in CD patients and HS group. We also analyzed the diagnostic utilities of all variables with significant differences between two groups.

We demonstrated that CD14+CD163+ monocytes were significantly more frequent in celiac disease patients than healthy subjects. CD163 is a scavenger receptor that participates in the removal of necrotic/apoptotic cells, which is normally expressed on the surface of M2 monocytes/macrophages. ²² Accordingly, the CD14+CD163+ population of monocytes represents the M2 subtype, and our findings are in favor of expanding the M2 subpopulation of monocytes in CD patients, in accordance with a recent publication. ³⁰ Barilli et al. demonstrated that gluten peptides are not only capable of enhancing the M2‐like polarization among monocytes of CD patients, but also they could interestingly drive M2 polarization in normal monocytes. ³⁰ Although several other studies have effectively divided monocytes into different (mostly three) subsets according to the CD14 and CD16 markers, ³² the evaluation of CD163 as a major anti‐inflammatory surface marker could be beneficial in better understanding the nature of inflammatory autoimmune conditions.

In order to functionally confirm the activation status of monocytes, we evaluated the serum levels of a selected numbers of pro‐ and anti‐inflammatory cytokines among CD patients and HS group. We realized that IL‐10 and TNF‐α expressions were noticeably elevated among patients, while TGF‐β expression was markedly decreased. In accordance with our findings, the elevation of IL‐10 has been also reported in previous studies by Forsberg et al. ³³ , ³⁴ and Manavalan et al. ³⁵ Moreover, several IL‐10 polymorphisms have been associated with the susceptibility to CD, ³⁶ , ³⁷ which highlights the importance of this multifunctional cytokine ³⁸ in the immunopathogenesis of CD. In order to better understand the role of IL‐10 in the pathogenesis of CD, Hollon et al. ³⁹ revealed that gliadin increased the secretion of IL‐10 from intestinal biopsy explants of CD patients and individuals with non‐celiac gluten sensitivity. Similarly and in accordance with our findings, several studies have shown that TNF‐α plasma expression was increased in CD patients, ⁴⁰ and the activation of blood monocytes by gliadin peptides could also elevate TNF‐α expression in CD patients. ¹⁶ Various polymorphisms of TNF‐α, especially in the promoter region, have been associated with the increased risk of CD, ⁴⁰ , ⁴¹ highlighting the significance of TNF‐α in this manner. Moreover, and similar to our findings, IL‐12 was not significantly changed between CD patients and normal subjects in previous research publication. ⁴² , ⁴³ However, the expression of TGF‐β has been reported to be elevated in the sera and lamina propria of patients with celiac disease, ⁴⁴ , ⁴⁵ which is in accordance with our findings.

We also quantified the soluble forms of CD16 and CD163 markers in the sera of CD patients and normal subjects. CD16 and CD163 are released from the surface of the monocytes/macrophages by proteolysis in response to oxidative stress or inflammatory stimulation and exerts their immunoregulatory effects on distant cells, organs, or tissues. The significance of these secreted, solubilized molecules has been addressed in several previous studies addressing autoimmune diseases, ⁴⁶ , ⁴⁷ , ⁴⁸ metabolic disorders, ⁴⁹ infectious diseases, ⁵⁰ , ⁵¹ and malignancies. ⁵² They have been also regarded as sensitive and specific biomarkers in the diagnosis of some disorders, ⁵³ increasing the importance of studying these molecules among CD patients. Daly et al. ⁵⁴ showed that the levels of sCD163 in untreated CD patients were higher compared with the treated patients, the disease control counterparts, and the healthy controls, while assessing sCD163 may be a useful method of monitoring the inflammatory lesions of CD. Similarly, Beitnes et al. ⁵⁵ found that the density of CD163(+) CD11c(+) APCs was increased in CD lesions. Sonier et al. ⁵⁶ revealed that finding putative autoantibodies against tolerogenic intestinal CD163+ macrophages could suggest that regulatory macrophages are targeted in CD patients. However and to the best of our knowledge, the serum levels of these molecules and their diagnostic values are being reported for the first time in the current study. Here, we showed that sCD16 serum levels were significantly elevated in CD patients.

In order to better characterize the polarization status of monocytes, CD163+/CD16+, IL‐10/IL‐12, TGFβ/TNFα, and sCD16/sCD163 ratios were defined and compared between CDPs and HS. It has been reported that these ratios, comprising of a major M1 and a main M2 marker in the same level, could better clarify the polarization status of monocytes/macrophages. ¹⁴ , ⁵⁷ Here, we showed that CD163+/CD16+ and IL‐10/IL‐12 ratios were significantly higher in CD patients, which are in favor of expanding the M2 subpopulation of monocytes in this group. ³⁰ Although the ratio of TGFβ/TNFα was higher in healthy subjects, it could be justified by the extremely elevated levels of TNFα in the inflamed sera of CD patients, which is a major component in the immunopathogenesis of CD. ⁵⁸

We also assessed the diagnostic values of all variables with significant differences between two groups of CD patients and healthy subjects. Normally, the first step in the diagnosis of CD is the serological evaluation of antibodies against transglutaminase II (IgA or IgG). ⁵⁹ If serologic tests are positive, the second step in confirming the diagnosis is duodenal biopsies, which are extremely invasive and rather expensive, especially among children. ⁶⁰ Here, we realized that the serum levels of IL‐10 and TNF‐α, in addition to immunophenotyping the CD14+CD163+ population of monocytes and evaluating the IL‐10/IL‐12 ratio could be used as fair diagnostic biomarkers in CD. The current study was associated with some limitations including small sample size and lack of information about the detailed nutritional status of patients which might affect the immunopathogenesis of the disease. ⁶⁰ It has been proposed that estrogen‐related signaling pathways and menstrual cycle could alter the polarization status of myeloid cells including monocytes and macrophages in favor of M2 expansion. ⁶¹ According to the higher incidence of CD among female patients, in order to better compensate the role of sex hormones on monocyte polarization, blood samples were taken during the first week of follicular phase of menstrual cycle (between day 0 and 7), where estrogen and progesterone levels were low. ⁶² Although the results of the current study should be confirmed in further investigations due to the small sample size and lack of specific subgroups analyses, the introduction of novel molecular biomarkers in CD could be promising.

5. CONCLUSION

Here, we evaluated the polarization status of CD14+ monocytes by analyzing the expression of M1 (CD16) and M2 (CD163) markers, the secretion levels of the soluble forms of these markers (sCD16 and sCD163), and the serum levels of IL‐10, IL‐12, TGF‐β, and TNF‐α cytokines in CD patients and HS group. We also assessed the diagnostic values of all variables with significant differences between two groups. We realized that CD14+CD163+ monocytes were more frequent in celiac disease patients than healthy subjects, and M2 subpopulation of monocytes was expanded among patients. We also showed that sCD16 serum levels were elevated in CD patients. In order to functionally confirm the activation status of monocytes, we demonstrated that IL‐10 and TNF‐α expressions were elevated among patients, while TGF‐β expression was decreased. In order to better characterize the polarization status of monocytes, we showed that CD163+/CD16+ and IL‐10/IL‐12 ratios were higher in CD patients, which were in favor of expanding the M2 subpopulation of monocytes. Finally, we showed that the serum levels of IL‐10 and TNF‐α, in addition to immunophenotyping the CD14+CD163+ population of monocytes and evaluating the IL‐10/IL‐12 ratio, could be used as fair diagnostic biomarkers in CD. The emergence of novel molecular biomarkers in celiac disease, especially the immunopathogenesis‐specific markers, could be promising in introducing novel diagnostic biomarkers for CD.

CONFLICT OF INTEREST

None.

AUTHOR CONTRIBUTIONS

OB participated in literature bibliography, acquisition of data, analyses and interpretations of data, manuscript drafting, and revision of the manuscript. EY participated in data acquisition and manuscript drafting. NA participated in data analysis and manuscript drafting. AS participated in statistical data analyses and manuscript drafting. FSSH participated in manuscript drafting. SM participated in literature bibliography, acquisition of data, analyses and interpretations of data, manuscript drafting, and revision of the manuscript. YY involved in study design and concept, literature bibliography, acquisition of data, analyses and interpretations of data, manuscript drafting, and revision of the manuscript.

RESEARCH INVOLVING HUMAN PARTICIPANTS

The present study which involved human participants was approved by the ethical Committee of GoUMS (Code of Ethics: IR.GOUMS.REC.1398.181). A written informed consent following the declaration of Helsinki was signed by all participators.

Babania O, Mohammadi S, Yaghoubi E, et al. The expansion of CD14+ CD163+ subpopulation of monocytes and myeloid cells‐associated cytokine imbalance; candidate diagnostic biomarkers for celiac disease (CD). J Clin Lab Anal. 2021;35:e23984. 10.1002/jcla.23984

DATA AVAILABILITY STATEMENT

Data supporting the findings of this study will be available from the corresponding author upon request.