We report a profound decrease in responsiveness to LPS after a chronic exposure to U. parvum
. Expression of all the measured genes with the sole exception of TGFβ1 did not increase in the lung in response to LPS in fetal lambs chronically exposed to U. parvum
. Consistent with previous reports (21
), we observed augmentation of LPS responses in vitro
in monocytes from the acute U. parvum
+LPS group compared to the LPS group or controls. However, LPS responsiveness was blunted in monocytes from the chronic U. parvum
+LPS group. Endotoxin tolerance is now understood to be a complex re-programming of inflammatory and non-inflammatory cells to repeated exposures to bacterial products (35
). Although a large body of literature on endotoxin tolerance is derived from in vitro
studies, historical studies have reported endotoxin tolerance in patients recovering from typhoid fever (36
). Also, leukocytes from patients with sepsis display endotoxin tolerance (37
). Our findings differ from these observations in several respects: Similar to human chorioamnionitis, intraamniotic injection of U. parvum
causes local colonization of the organism in the organs contacting the amniotic fluid e.g. lung, chorioamnion, gut and the skin, but infrequently causes systemic bacteremia (10
). Secondly, we found a near global decreased capacity to respond to LPS in the fetal lung after a chronic U. parvum
exposure rather than a complex re-programming of inflammation. Thirdly, the down-regulated LPS responsiveness was demonstrated 70d after exposure but not after a 7d exposure. Our study is unique in that, the host is a preterm fetus with developmental immaturity of the immune system.
spp are common colonizers of the lower genital tract, there is some debate about the pathogenicity of the organism in pregnancy. However, several studies have unequivocally demonstrated the association of female upper genital ureaplasma colonization with preterm deliveries, fetal inflammation and adverse neonatal outcomes (8
). Furthermore, Ureaplasma
species can induce inflammatory cells to produce pro-inflammatory cytokines via TLR2/6 in vitro
). Consistent with its low virulence, we observed a persistent colonization but low-grade lung inflammation both at 7d and 70d after intra-amniotic injection of U. parvum
. Lung inflammation after intraamniotic Ureaplasma exposure is likely via direct colonization, since we previously reported a 2-log order higher Ureaplasma titer in the fetal lung fluid compared to the amniotic fluid (14
). Ureaplasmas are known to produce biofilms, which could trap inflammatory products thereby mechanically block inflammatory signaling (38
). However, this is an unlikely explanation for the profound hypo responsiveness to LPS in our experiments, since LPS responses were preserved when U. parvum
exposure was for 7d. Further, both lung and blood monocytes from fetal lambs exposed to U. parvum
for 70d but not 7d demonstrated decreased responsiveness to LPS in vitro. Whether these responses are dependent on the degree of prematurity is not known. Also, the precise mechanism/s by which a chronic exposure to U. parvum
can decrease LPS responses remain to be identified.
Exposure to chronic but not acute U. parvum infection decreased both the influx of pulmonary inflammatory cells and expression of activation markers induced by intraamniotic LPS. Therefore, a question is whether the decreased expression of activation markers reflected decreased inflammatory cells in the fetal lung. The inflammatory cell composition in the lung 2 days after intraamniotic LPS is roughly 60% neutrophils and 35% monocytes with few lymphocytes. Compared to effects of LPS alone, a prior exposure to chronic U. parvum reduced the neutrophil influx by about 30% and monocytes by 60% (these reductions were not statistically significant). In contrast, the reductions in expression of cytokines, PU.1+, MCP-1+, and MPO+ cells were much larger, and the monocytes from fetal lambs with chronic U. parvum + LPS were poorly responsive to an in vitro LPS challenge. Taken together, these findings suggest that chronic exposure to U. parvum decreased activation and possibly recruitment of leukocytes to the fetal lung in response to LPS.
Endotoxin tolerance causes a complex re-programming of inflammatory responses (35
). Pro-inflammatory cytokine expression is down regulated, while there is no change or even an increase in the expression of anti-inflammatory genes, antimicrobial genes, genes mediating phagocytosis (35
). Indeed, microarray analysis of tolerant vs. non-tolerant monocytes demonstrate a host of genes that are down regulated, while other genes are not down regulated (39
). The net result of endotoxin tolerance appears to prevent host organ injury while maintaining antimicrobial functions. However, almost all the information regarding endotoxin tolerance is derived from gene expression in functionally mature monocytes or macrophages. Since endotoxin tolerance is an adaptive host response, the characteristics of endotoxin tolerance might vary depending on the inflammatory context. We previously reported that preterm sheep fetuses exposed to repeated doses of intraamniotic LPS demonstrate endotoxin tolerance and cross-tolerance to other toll-like agonists (19
). The genes reported not to be down regulated after endotoxin tolerance include IL-10, IL-1ra, TGFβ1, serum amyloid and others (39
). While IL-10 and IL-1ra were also down regulated in the chronic U. parvum +LPS animals in our study, TGFβ1 increased relative to controls. Another class of genes not down regulated during endotoxin tolerance is the TRIF mediated interferon inducible genes (40
). Further, interferon signaling negatively regulates TLR4 signaling and can abrogate endotoxin tolerance in blood monocytes (41
). In the present experiment however, the pattern of expression of the interferon inducible genes CXCL9 (MIG) and CXCL10 (IP10) mRNA expression in the lung after chronic U. parvum
+ LPS exposure was similar to the other pro-inflammatory cytokines. These results suggest that chronic exposure to U. parvum
induced down regulation of LPS responses were more global in nature than previously reported in endotoxin tolerance.
Both intracellular negative regulators and extracellular soluble factors have been implicated in the mechanism of endotoxin tolerance. Extracellular/humoral factors that potentially mediate endotoxin signaling include steroid hormones (43
), HSP-70 (44
), and IL-10 (45
). The plasma cortisol levels (data not shown) and lung expression of IL-10 mRNA did not increase in these fetal lambs. However, TGFβ1 expression in the airways was higher in the lambs with down-regulated LPS responses compared to controls. These data are consistent with the known anti-inflammatory properties of TGFβ1 (32
). We previously reported increased expression of TGFβ1 and the downstream mediator phospho-SMAD2 after exposure to intraamniotic LPS (46
). Both TGFβ1 and p-SMAD2 are detected in multiple lung cell types in the epithelium and the interstitium of fetal lung. The expression of mRNAs for the receptor TLR4, or the downstream transcription factors IRAK-1, IRAK-4, or IRAK-M in the lung did not change. Similarly, a growing list of intracellular mediators including MyD88s (47
), IRAK-M (34
), SIGIRR (48
), SOCS-1 (49
) and others have been proposed as mediators of endotoxin tolerance. We previously reported that TLR4 mRNA is expressed ubiquitously in most of the lung cell types in the preterm fetus (50
). In the absence of cell-type expression data for downstream mediators of TLR signaling, a caveat in the interpretation of changes in expression is that whole lung homogenates used for quantification of mRNAs may dilute the changes in the relatively non-abundant recruited inflammatory cells.
Regulatory T-cells (Tregs) expressing FOXP3 are abundant in the fetal circulation and down regulate inflammatory responses (51
). However in our study, acute or chronic exposure to U. parvum
did not change FOXP3+ cells in the mediastinal lymph node or the lung (data not shown). Another mechanism for endotoxin tolerance is chromatin remodeling, changes in histone acetylation and methylation induced gene silencing (39
). While the precise molecular pathways of down regulated endotoxin responses in chorioamnionitis remain to be determined, increased TGFβ1 expression was demonstrated in the lungs of fetal lambs with down-regulated LPS responses.
In contrast to sepsis, exposure of the fetus to bacterial components in chorioamnionitis is via the epithelia of the airway, the chorioamnion and the gastrointestinal tract but not the vascular compartment. The resulting systemic inflammatory response is a mild increase in cytokines and acute phase reactants rather than the cytokine storm associated with sepsis in the adult (52
). Liver expression of acute phase reactant genes CRP and serum amyloid A3 and plasma haptoglobin increased after intraamniotic LPS as expected, but the expression was not down regulated in lambs exposed to chronic U. parvum
+LPS. These findings suggest that liver, the major source of acute-phase reactants, was not subject to down-regulated LPS responses after exposure to chronic U. parvum
. However the blood monocytes from these chronic U. parvum + LPS animals were poorly responsive to LPS. Taken together, these findings suggest that LPS responsiveness in the fetus differed in different organs.
There are several clinical implications of our findings. Although Ureaplasma
spp are the organisms most commonly associated with chorioamnionitis, about 60% of cases with chorioamnionitis have co-infection with ureaplasma and other microorganisms (3
). Our findings suggest the possibility of decreased fetal inflammation in response to Gram-negative organisms with concomitant ureaplasma exposure. On the other hand, analogous to adults with sepsis (37
), diminished innate immune responses could cause adverse fetal outcomes. Similarly, post natal nosocomial sepsis occurs in about 25% of very low birth weight preterm neonates (54
). Our data suggests the possibility that ureaplasma chorioamnionitis could diminish innate immune responses and thereby increase the susceptibility of preterm infant to nosocomial sepsis. In summary, the experiments demonstrate a novel finding that a chronic exposure to intraamniotic U. parvum
decreases both in vivo
and in vitro
lung endotoxin responsiveness in a preterm fetus.