Assessment of peripheral blood lymphocyte subsets in children with iron deficiency anemia

Iron deficiency anemia is the most common preventable nutritional deficiency [1].

Iron and immunity are found to be in a close relationship. Iron utilization by invasive bacteria is controlled by genes/proteins which regulate iron flux to such organisms thus interfering with their growth. Moreover, through hepcidin and ferropotin, iron flux into the bacterial cell is controlled and this is mediated by the cells of innate immune system e.g. Monocytes, macrophages, and lymphocyte cells of the innate immune system. In addition, many effector molecules, e.g. haem oxygenase, toll-like receptors, hypoxia factor-1, NF-κB which are the regulatory molecules in the inflammatory response organize a multiplicity of cytokines, chemokines, reactive oxygen and nitrogen species at which either iron loading or depletion occur. This may adversely affect the ability of the cell to respond to the bacterial insult [2].

Iron has an essential role in surveillance of immune cells, particularly in lymphocytes, due to its growth-stimulating and differentiating properties [3]. Also, iron is necessary for monocyte/macrophage differentiation [4]. However, humeral immunity seems to be less affected by iron deficiency compared to cellular immunity [5].

Iron is also critical for enzymes involved in DNA synthesis, and the proliferative phase of lymphocyte activation is a Fe⁻demanding phase and it can be weakened during IDA [6] thus results in altered expression of cell markers that may contribute to the reduced T-cell proliferation [7].

However; there are three types of mature T cells based on their surface receptor expression; T helper cells (CD3+/CD4+), T-suppressor/cytotoxic cells (CD3+/CD8+) and γ/δ T cell (CD2+, CD3+, CD7+, CD4− and CD8−) that function as a type of cytotoxic cells [8].

Data on the effect of iron deficiency on immune function are confusing and contradictory. We aimed to assess the percentage and absolute counts of the peripheral blood lymphocyte subsets in children with iron deficiency anemia by a flow cytometric assay and correlate the results with the clinical and laboratory criteria of the affected children.

Seventy-five children were enrolled in this study, 50 patients with confirmed iron deficiency anemia (IDA) (their median age was 8 years). Twenty-three were males and 27 were females. Another healthy 25 age and sex-matched children were enrolled as controls (12 were males and 13 were females and the median age was 7.2 years (Table 1).

Patients had significantly lower hemoglobin concentration, Serum iron (p = 0.001 for all), ferritin and higher lymphocytic count (p = 0.03) in IDA patients compared to the controls. There was no significant difference between the two groups regarding white blood cell count (WBC), neutrophil count, neutrophils/lymphocyte ratio and platelets count (Table 2).

IDA patients showed a significant reduction in the count and the percentage of CD4 compared with controls (p = 0.001 for both). No significant difference between both study groups regarding CD8 count and percentage (p = 0.22 and 0.71 respectively). The CD4/CD8 ratio was significantly lower in IDA patients (p = 0.005). No significant difference between the two studied groups regarding either CD19 count or percentage (p = 0.28 and 0.18 respectively) (Table 3).

Significant positive correlation was found between serum iron level and absolute neutrophil count (p = 0.02, r = 0.27), CD4% (p = 0.001, r = 0.5) and CD4/CD8 ratio (p = 0.002, r = 0.35) while significant negative correlation was found between serum iron level and CD19 count (p = 0.02, r = 0.27) (Table 4).

Cellular and humoral immunities are mediated by lymphocytes (T and B) respectively. T lymphocytes are formed by the thymus gland and are released into the peripheral blood constituting more than 60% of the whole lymphocytic count [8]. Two types of mature T lymphocytes are formed. The T helper cells which express CD3+/CD4+ cell markers and T suppressor/cytotoxic cells which express CD3+/CD8+ cell markers. Mature B lymphocytes, however, are formed in bone marrow and express CD19 cell marker.

Iron was confirmed to be a vital element for immune system development and play an important role in the integrity of the immune system [9‐11]. In the present study, IDA patients had a significant decrease in the total lymphocyte count compared to controls. Regarding lymphocytes subpopulation, T-lymphocytes (patients had a significantly lower number and percentage of CD3 positive cells) were significantly decreased while B lymphocytes (presented by CD19 positive cells) showed no significant difference compared with healthy children. This comes in agreement with the results of Kuvibidila et al., [12] who confirmed that T lymphocytes are much affected with iron than B lymphocytes.

Regarding T-lymphocyte subsets, the percentage and the absolute count of T helper cells (CD4 positive) were significantly lower compared to controls. Omara and Blakley, [13] proved a lower delayed hypersensitivity response (where CD4+ lymphocytes are the key marker) and decreased lymph proliferative activities in mice fed an iron-deficient diet than those fed a normal or supplemented iron diet. These results suggested impaired T-helper cell response in iron deficiency anemia [14].

The T-lymphocyte inhibition mechanisms in IDA patients are not fully understood. CD4+ lymphocytes are affected by iron deficiency which prevents the development of immune response against different pathologic challenges [15]. The function of different enzymes involved in cell-cycle control, such as the ribonucleotide reductase (involved in DNA synthesis during cell cycle S phase) is depressed by Intracellular iron deprivation [16, 17]. Iron restriction can also inhibit phosphatidylinositol-4, 5-biphosphate hydrolysis, and kinase C protein activity, both of which are important steps in the intracellular signaling cascade which is initiated by T cell activation [12]. Saldanha-Araujo and Souza, [18] and Markel et al., [19] reported that T lymphocytes iron uptake and their cellular proliferation is altered in IDA. Furthermore, the proliferation of T helper lymphocytes as Th-1 is much sensitive to the changes in the iron levels compared to the Th-2 lymphocyte. This is attributed to the difference in transferrin-related iron uptake [19].

Our results showed insignificant alteration in the percentage and the absolute count of CD8 positive lymphocytes. This is in agreement with Das et al., [20] and Mullick et al., [21, 22]. While Thibault et al., [23] found a decreased in the IL-2 production in IDA patients which was explained by alteration in CD8 positive lymphocyte maturation.

Iron was found to be a vital component of peroxide and nitrous oxide–generating enzymes. These enzymes are required for the proper immune cell function. Also, iron is involved in the regulation of cytokine production through its influence on second-messenger systems [24].

Phytohemagglutinin-induced lymphocyte proliferation and delayed-type hypersensitivity responses were found to be reduced in nutritional iron deficiency with relative preservation of humoral immunity [17]. Moreover, iron deficiency in humans can alter the cytokine expression profile of the activated lymphocytes [16].

The altered levels of some interleukins (IL) and cytokines (e.g. IL-2, IL-1, IL-6, TNF-α, IL-4, IL-12p40, IFN-γ, and IL-10) might result in immune system impairment in IDA patients [25, 26]. Also, altered cell marker expression may contribute to reduced T cell proliferation during iron deficiency [12].

Galan and colleagues, [27] reported a reduction in interleukin-2 production by activated lymphocytes in iron-deficient subjects. The release of interleukin-2 is required for communication between lymphocyte subsets and natural killer cells but it does not appear to be the only cytokine that is altered by iron status [15].

Regarding anti-microbial host responses, Iron plays important roles first by synergistic anti-microbial radical formation action [28‐30] and second, by direct alteration of both immune cell proliferation and anti-microbial immune effector pathways [31]. Thus, the host immune system affects the availability of iron for microbes via the activity of cytokines.

In our study, the CD4: CD8 ratio was lower in children with IDA. This was in agreement with Das et al., [20] and Mullick et al., [21, 26]. Iron deficiency alters the proportion and function of various T cell subsets. Occasionally, the total lymphocyte count in the peripheral blood is low. All these changes are reversed by iron therapy. An altered immune response after iron administration may indicate the existence of an unsuspected functional iron deficiency. Several previous studies confirmed that iron supplementation improved the different subsets of the mature T-cell count and this is agreeing with the results of Sejas et al., [32] who confirmed that iron supplementation in children can significantly accelerate proliferation and maturation of the circulating immature lymphocyte subpopulations [33].

Some limitations had faced our study. Due to cultural and traditional issues, a lot of patients refused to be included in the study which led to a small sample size of the study.

Iron deficiency anemia (IDA) in children can alter the lymphocyte subset in the absence of other factors challenging the immune system. Studies involving a larger number of children, belonging to well-defined age groups from different geographic areas are recommended for conclusive interpretation in the future.