Early Pregnancy Regulates Expression of IkappaB Family in Ovine Spleen and Lymph Nodes

**Background:** Early pregnancy requires modulation of the maternal immune system to prevent rejection of the allogenic conceptus while maintaining immune competence. Nuclear factor kappa B (NF-κB) is critical in both physiological and pathological processes, and its dysregulation can lead to pregnancy complications. The IκB family proteins are inhibitors of NF-κB that regulate its activity. Previous studies have shown that early pregnancy modulates NF-κB components in various maternal tissues in sheep, but the specific expression patterns of IκB family members in the spleen and lymph nodes during early pregnancy were not well characterized.

**Methods:** Mature Small-tail Han ewes were divided into four groups (n=6 each): non-pregnant ewes at day 16 of the estrous cycle, and pregnant ewes at days 13, 16, and 25 of gestation. Spleens and inguinal lymph nodes were collected. Quantitative real-time PCR (qRT-PCR) was used to measure mRNA expression of BCL-3, NFKBIA (IκBα), NFKBIB (IκBβ), NFKBIE (IκBε), IKBKG (IKKγ), NFKBID (IκBNS), and NFKBIZ (IκBζ). Western blot analysis was performed to assess protein levels of these IκB family members. Immunohistochemistry was used to localize IκBβ and IKKγ proteins in tissue sections. Data were analyzed using the MIXED procedure in SAS with Duncan's method for multiple comparisons.

**Key Results:** In the spleen, early pregnancy induced expression of BCL-3, IκBα, IκBε, IKKγ, and IκBζ. BCL-3, IκBβ, and IκBNS peaked at day 16 of pregnancy (p<0.05). IκBα, IκBε, IKKγ, and IκBζ proteins gradually increased from day 13 to 25 of pregnancy (p<0.05). In the lymph nodes, early pregnancy suppressed BCL-3 and IκBNS expression but stimulated IκBβ and IκBζ expression. IκBα, IκBβ, IκBε, and IKKγ peaked at days 13 and/or 16 of pregnancy (p<0.05). Immunohistochemistry showed IκBβ and IKKγ localized to the capsule, trabeculae, and splenic cords in the spleen, and to the subcapsular sinus and lymph sinuses in lymph nodes. Staining intensities varied across pregnancy stages, with the strongest IκBβ staining (score 3) observed at day 16 of pregnancy in both spleen and lymph nodes.

**Clinical Implications:** The tissue-specific regulation of IκB family members during early pregnancy suggests that these proteins play distinct roles in modulating immune function in the spleen and lymph nodes. The upregulation of BCL-3, IκBα, IκBε, IKKγ, and IκBζ in the spleen may contribute to splenic adaptive immunity and B cell maturation. In lymph nodes, the suppression of BCL-3 and IκBNS alongside stimulation of IκBβ and IκBζ may be related to establishing maternal immune tolerance. These changes are likely mediated by conceptus-derived interferon-tau (IFNT) and progesterone from the corpus luteum. Understanding these mechanisms could provide insights into pregnancy maintenance and immune tolerance, with potential relevance to pregnancy complications such as pre-eclampsia and preterm birth. However, as this is an animal study in sheep, direct translation to human pregnancy requires further investigation.