Although there exist several proposed mechanisms to explain PTD, and several soluble and membrane-bound mediators are thought to play significant roles, it is not yet clear which cells or cellular circuits are critical to the initiation, completion, or regulation of this process.
By examining Rag1−/−
mice, we first tried to understand the critical importance of T and B cells. In our study, Rag1−/−
mice delivered 24 h after LPS injection, which implies that the adaptive immune system is not critical for LPS-induced PTD. We also observed an increase in proinflammatory cytokines in the serum after injection of LPS, which is consistent with the findings by other groups studying LPS-induced PTD in normal mice [13
]. The inflammatory cytokine response that we observed in Rag1−/−
mice included an increase in mediators that activate and attract neutrophils and macrophages, and these, along with NK [21
] cells, would be expected to be the primary cellular positive regulators of LPS-induced PTD in Rag1−/−
mice. After injection of LPS, we also observed an increase of RNA expression of key molecules, such as MyD88 and NFkappaB, thought to be critical downstream signaling molecules in the pathway linking TLR4 and PTD [43
]. We also observed an increase in PTGS2 (COX2) expression, possibly by uterine-resident immune cells, which can support local levels of prostaglandins and, in turn, uterine contractions. With the caveat that expression at the protein level may be different from that at the RNA level, pregnant Rag1−/−
mice exhibit activation of the same pathway(s) in response to LPS as do mice with working adaptive immune systems.
These data, along with those of others [21
], underscore the importance of the innate immune response in LPS-induced PTD, and in other models of pregnancy loss [22
Although the increase in sensitivity to low doses of LPS that we observed in Rag1−/−
mice may relate to inherent reproductive dysregulation in the absence of T and B cells, we did not observe evidence to support this, as, for example, our Rag1−/−
mice did not experience a higher rate of early loss (data not shown) compared to normal mice. To explain this difference in sensitivity, we instead hypothesized that T and B cells would serve a regulatory role in this process. This hypothesis was supported by the results obtained after transfer of whole-lymphocyte preparations from normal into Rag1−/−
mice. In light of current interest in the role of regulatory T cells in immune responses, we examined the effect of reconstitution of Rag1−/−
mice with cells significantly enriched (>90% pure) in CD4+
T cells. While preinjection with this cellular preparation suggests a regulatory role for CD4+
T cells, it does not rule out a role for “contaminating” accessory cells or B-1 B cells [40
] in regulation of this response. Moreover, these studies do not shed light on whether this regulation is on the basis of soluble mediators (e.g., cytokines), or direct cell-cell contact. Finally, these experiments do not rule out a role for CD8-expressing T cells with regulatory function.
Even if pure, CD4+
T cells comprise a heterogeneous population of naïve, effector, and memory cells, as well as cells with true modulatory or down-regulatory function, and this may be the reason why the LPS response in CD4+
T cell-reconstituted Rag1−/−
mice was not completely suppressed. However, it is also clear that “regulatory T cells” also denotes a heterogeneous family of cells, potentially with differing phenotype and functional capacity. A more prominently considered subgroup of regulatory T cells expresses FOXP3 [42
], and the role of this cell type in immune regulation is under intense investigation in vivo in manipulated and intact animals. Earlier studies delineated regulatory T cells as CD4+
, and have suggested a regulatory role for cells carrying this phenotype in models of recurrent pregnancy loss [27
] and in normal allogeneic pregnancy [25
]. Current thinking, moreover, is that the majority of CD4+
regulatory T cells also express FOXP3. Our finding of CD4+
cells in Rag1−/−
mice relatively resistant to LPS-induced PTD after pretreatment with CD4 T cells suggests that so-defined regulatory T cells may be a component of the existing protective cellular circuit. It is also formally possible that, in our model, as has been described in other systems [45
], this protective circuit also comprises regulatory T cells that posses key regulatory functions, but do not fit the currently accepted definitions. Finally, in this model, the idea of regulatory T cells, not as a separate lineage, but more broadly as a subset expressing cytokines (e.g., IL10) that oppose PTD, needs to be considered.
PTD represents a complex biological process that is currently difficult to accurately diagnose until its final stages, involving active labor and imminent delivery, become evident. However, as our understanding of the critical regulators and intermediate steps involved in PTD increases, potential targets for both diagnostic tests and therapeutic intervention will become apparent. The studies in this report suggest that focus on cellular components of the innate immune response activated during LPS-induced PTD may identify new diagnostic tools. Moreover, focus on related regulatory circuit(s) involving T cells may support development of novel interventions.