Characterization of Maternal Macrophage Responses to Placental Malaria Among Pregnant Women Attending Antenatal Clinic in Webuye County Hospital, Bungoma, Western Kenya

Abstract

BACKGROUND: Decidual macrophages are essential during pregnancy playing the most critical role in tolerance, tissue priming, parturition, and postpartum recapability. Change in the placental microenvironment leading to altered polarization state causes major pregnancy-related deleterious effects. Malaria, a highly inflammatory disease, occurs in the placenta and is likely to tip the balance of the tightly regulated immunological response associated with pregnancy. Macrophages are the second most abundant immune cells in the placenta, following uterine natural killer cells (uNK), and they play a vital role in maintaining pregnancy. These macrophages are categorized into two types: classical (M1) and alternative (M2). M1-like macrophages are known as proinflammatory macrophages due to their protective role, while M2 macrophages are classified as anti-inflammatory because they mainly handle tissue repair and remodeling. In addition to these functions, macrophages are involved in several other specific processes essential for pregnancy maintenance. This study determined the distribution of M1 and M2 macrophage responses in placentas from infected versus uninfected participants, assessed the expression of transcription and angiogenic factors involved in M1 and M2 pathways, and determined how their pattern in placentas from malariainfected versus uninfected, correlated with M1/M2 cytokine production with Th1/Th2. METHODS: The study enrolled women aged 18 to 45 (n=60) attending antenatal clinics in Webuye County Hospital, Bungoma, Western Kenya. Upon delivery, placentas, and dry blood spots (DBS) of placental blood were collected. DBS was used for malaria diagnosis. Following collagenase digestion, placental samples were used as the source of macrophages and other immune cells for downstream characterization. The recovered immune cells were analyzed by flow cytometry for surface markers including CD68, CD80, CD86, CD163, CD206, and CD209. For gene expression analysis, macrophages were isolated from the immune cells using anti-CD14 beads, and the levels of transcription and angiogenic factors were analyzed by qRT-PCR (targeting STAT-1, IRF5, STAT-6, cMAF, ANG-1, and ANG-2). This was concluded by stimulating both T cells and macrophages for cytokine expression using PMA-ionomycin and LPS respectively to understand the functionality of these cells. RESULTS: Sixty pregnant mothers were enrolled in this study. Out of this, 26 (44.07%). were malaria-positive by histology while 27 were positive by PCR (46.55%). The highest proportion of pregnant mothers were those aged between 20 to 35 (71.20%) with primigravid accounting for 39%. While malaria was associated with younger mothers (P=0.037). Low birth weight was record in 11.90% of the newborns. M2 macrophages were significantly expressed in the placentas in all groups (women with and without malaria) (P=0.0001). However, no statistically significant differences were observed for macrophage markers (CD68, P=0.5586, CD80, P=0.7282, CD86, P=0.9641, CD206, P=0.9122 and + and CD163/CD206, P=0.4441). Notably, the gene expression levels of angiogenic factors ANG-1 (P=0.0396) and M2 transcription factor STAT-6 (P=0.0116) were significantly higher in the malaria-negative population as others were insignificant (STAT-1, P=0.0908, IRF-5, P=0.0560, cMAF; P=0.0689). While there were no significant differences observed in the cytokines expression between the two groups, an upward trend in TNFα (P=0.0457) was observed in malaria positive group. Interestingly, there was reduced expression of the IL-10 and IL-4. P < 0.05 was considered statistically significant. CONCLUSION: The findings of this study highlight the crucial involvement of placental macrophages in malaria infection, shedding light on previously overlooked immune signature dysregulation and angiogenesis in placental malaria. This study has shown that decreased expression of ANG-1 is indicative of placental malaria (PM), while reduced expression of STAT-6 in individuals with malaria indicates improved protection against PM, supporting a healthy pregnancy. The downregulation of ANG-1 and STAT-6 expression suggests the inhibition of anti-inflammatory markers, evidence of increased pro-inflammatory as shown by increased TNFα implying skewness to malaria infection status.