Milk Tumor Necrosis Factor Alpha and Interleukin-1Beta Among Toxoplasma gondii-Free and Infected Women

Pro-inflammatory cytokines play an important role in intercellular communications. In the last two decades, many cytokines have been identified in human milk. These cytokines are variable according to different conditions such as pathogenic infections which strongly stimulated the immune response. The present study aims to determine of IL1β and TNF-α in Toxoplasma gondii-free and infected women in an attempt to clarify the impacts of the infections on cytokines especially in mother's milk. The serum and milk sample were collected from 96 samples (48 for seropositive and 48 for seronegative). To confirm the Toxoplasma gondii infection; enzyme linked immunofluorescence assay (ELIFA) was used to detect antiToxoplasma IgG antibodies. TNF-α and IL1β levels were measured by ELISA technique. Regarding milk TNF-alpha, there were statistical differences in Toxoplasma-infected from Toxoplasma-free subjects, whereas no statistical variations in TNF-α were found between other studied groups. IL1β showed significant variation just between milk and serum in infected and non-infected subjects, whereas no variations were recorded in other groups. In conclusion, based on statistical analysis, present study showed good evidence that toxoplasmosis is one of the risk factors for increasing milk cytokine. Also, a significant increase in TNFα and IL1β in mother's breast milk may indicate a key role for inducing or developing of inflammations in suckling. Thus, further investigations are needed to cover this area of study.


Introduction:
In the last two decades, different cytokines have been identified in breast milk. Most of these cytokines come from epithelial cells of mammary tissues, local immune cells and the mother's circulation system 1 . Maternal breast milk cytokines have potential impacts on the newborn immune system. Milk is not only a source of feeding but also as a method of adapting infants to their extra-uterine environment 2 . Immune cells in the mother's body play an important role to prevent fetal rejection. As well-known, pregnancy is controlled by having different concentrations hormones, such as estrogen and progesterone 3 . In addition, cytokines perform an important role in the success of pregnancy 4 . Several studies showed that the factors associated with abortion are Th1 while Th2 which are the factors that help to sustain pregnancy 5 . Further studies also indicate that normal pregnancy is related with an improved immune response to Th2 while abortion occurs to raise the immune response to Th1. IL-12 has a crucial role in fetal loss stimulates natural killer cells output and TNF-α by macrophages and T lymphocytes 6 . The network of cytokine mediator in milk has not been wellstudied, especially in case of parasite infections. As well known the T. gondii is the most successful parasite with prevalence rate from 5 to 90 % 7 . T. gondii invade different tissues such as brain, heart, lung, lymphoid and recently it's found in breastmilk 8 . Many researchers have discussed the roles of cytokine networks for the development and progression of many diseases, and this production of cytokines is almost related to invasion of pathogens especially parasitic infections 9 . Therefore, in the light of increasing inflammatory diseases, the effects of parasites invasions on cytokines variability may give us an indication of this relationship.

Subjects:
All exclusion criteria, such as family history, chronic and genetic disease, drinking and smoking were excluded in current study. Ninety-six enrolled women had been complete clinical data and bioinvestigation for the subdivision into two groups, Toxoplasma-free group and, Toxoplasma-infected group.
Blood and milk collection Five mls of milk were collected from enrolled women after4-6 weeks postpartum, then putted in sterilized tubes and stored in the refrigerator, whereas blood was transferred to the gel tube. Serum was separated from whole blood by centrifugation (4000 rpm for 5 min.) and then blood was stored in the refrigerator immediately at -20 0 C.

Determination of Toxoplasmosis:
Serum anti-toxoplasma IgG antibodies were identified by using immunofluorescence assay (IFA). Briefly, this assay principle combines an enzyme immunoassay method by immunocapture with final fluorescent detection (ELFA). The procedure of this assay was accomplished by manufacture commercial kit by Biomerieux Company (France).

Measurement of TNF-α and IL1β
The concentration of TNF-α and IL1β was measured by using Enzyme-Linked Immunosorbent Assay (ELISA) technique. Commercial ELISA kit from Elabscience Company was used for this purpose.

The study protocol and ethics
The study protocol was approved by the local ethical committee in the college of science / Babylon University, declaration Helsinki standards were adopted in the present study.

Statistical Analysis
Statistical analyses were performed using a statistical software program (SPSS 10 Inc., Chicago, USA). T-test was used to search for differences regarding the nominal variables in our study. Variations were considered significant when P-value ≤ 0.05

Results:
Results of milk TNF-alpha in Toxoplasma gondii in free and infected women are shown in Fig. 1. The differences in the median values between the two groups are greater than that would be expected by chance; thus statistically significant difference existed (P = 0.018). TNF-alpha concentration was significantly higher in milk Toxoplasma-in the infected women as compared with non-infected subjects. No statistically significant difference was found between serum TNF-α levels in women with the Toxoplasma seropositivity group in comparison with those women who have seronegative ( Fig. 1, P = 0.168). Furthermore, TNF-α levels variations in the median values between milk and serum in seronegative subjects are not great enough to exclude the possibility (P = 0.128). Finally, the TNF-α levels did not reach the significant variation between milk and serum in Toxoplasma-free and Toxoplasma-infected women Fig. 2. (P= 0.053). IL1β showed significant variation between milk and serum in infected and non-infected subjects with values of 0.035 and 0.003 respectively (Figs. 3, 4). Also, our results showed no variations between IL-1beta in seropositive and seronegative subjects (Fig.  4, P=8). The data of IL-1beta levels in Toxoplasmafree and infected women showed no significant variation (Fig. 3, P-value=0.128). Personal correlations test showed significant variations between serum TNF-α and IL1β in seropositive subjects (P-value=0.000267). Also, a positive correlation is shown between milk TNF-α and IL1β in seropositive patients (P-value=0.000293).  In the present study two pro-inflammatory cytokines (TNF-α and IL1β) were investigated in women during the first six weeks of lactation. It is well-known that IL1β and TNF-α are macrophage stimulating cytokines in which both mediates inflammation and shares many systemic responses, development, growth, hematopoiesis and cell signaling 10,11 . Data presented here showed significant differences in milk TNF-α level (Fig.1). Also, the same variations were found in milk and serum IL1β levels among Toxoplasma-free and infected women (Fig.4). Most of the inflammatory cytokines, such as TNF-α, IL-1β, IL-6, IL-8 and IFN-γ are found at lower concentrations compared to anti-inflammatory cytokines 12 . Also, these concentrations decrease over lactation 13 .

Open
However, during parasite infection the progress of pregnancy and mother's immune system response may have a different story 14 . Such responses may reflect strategies of host and parasites to maximize the fitness for each other. Also, it could be stated that milk is a communication tool be for mother-infant for protection against pathogens. Previous studies were suggested that the incidence of T. gondii leads to increase serum pro and anti-inflammatory cytokines levels 15 . Although, increasing the production of cytokines is associated with the protection against parasitic infections, yet this mechanism may be costly because the over cytokine productions induces another disease in mothers and infants 16,17 .
Furthermore, other studies suggest that abortion is always associated with increasing TNFα level 12 . Therefore, the increase in cytokines productions may be related with parasite strategies to increase a chance of abortion. Our findings also show an increase in TNF-α concentrations in breast milk of the infected mothers. These variations may be due to T.gondii infections that increase maternal injury or local cytokines production. Further, our results showed a positive significant correlation between TNF-α and IL-1β in the serum and milk of the infected mothers (Pearson correlations). This correlation confirms that the increasing levels of cytokines in the blood lead to the rise in milk. Cytokines increasing may be depicted the vital effects of toxoplasmosis in cytokine outcome and their effects on mother-infant pathological mediate. Also, cytokines outcome modulations may increase understanding the important role of pathogens in mother-infants regulation and development of immune response. Thus, further investigations are needed to cover this fruitful research area.

Conclusion:
The importance of the current findings especially those related to increase proinflammatory cytokine in the toxoplasma-infected mother's milk makes the first step for shifting the understanding of the pivotal roles of cytokines, TNF-α and IL-1β, in pregnancy gaining or losing as well as the inductions or protections of many diseases in infants.
the Figures and images, which are not mine ours, have been given the permission for republication attached with the manuscript. -The author has signed an animal welfare statement. -Ethical Clearance: The project was approved by the local ethical committee in University of Babylon.