Class II obese and healthy pregnant controls exhibit indistinguishable pro‐ and anti‐inflammatory immune responses to Caesarian section

Abstract Introduction Obesity during pregnancy is associated with meta‐inflammation and an increased likelihood of clinical complications. Surgery results in intense, acute inflammatory responses in any individual. Because obese individuals exhibit constitutive inflammatory responses and high rates of Caesarian section, it is important to understand the impact of surgery in such populations. Whether more pronounced pro‐inflammatory cytokine responses and/or counterbalancing changes in anti‐inflammatory immune modulators occurs is unknown. Here we investigated innate immune capacity in vivo and in vitro in non‐obese, term‐pregnant controls versus healthy, term‐pregnant obese women (Class II, BMI 35–40). Methods Systemic in vivo induction of eleven pro‐ and anti‐inflammatory biomarkers and acute phase proteins was assessed in plasma immediately prior to and again following Caesarian section surgery. Independently, innate immune capacity was examined by stimulating freshly isolated PBMC in vitro with a panel of defined PRR‐ligands for TLR4, TLR8, TLR3, and RLR 24 h post‐surgery. Results The kinetics and magnitude of the in vivo inflammatory responses examined were indistinguishable in the two populations across the broad range of biomarkers examined, despite the fact that obese women had higher baseline inflammatory status. Deliberate in vitro stimulation with a range of PRR ligands also elicited pro‐ and anti‐inflammatory cytokine responses that were indistinguishable between control and obese mothers. Conclusions Acute in vivo innate immune responses to C‐section, as well as subsequent in vitro stimulation with a panel of microbial mimics, are not detectably altered in Class II obese women. The data argue that while Class II obesity is undesirable, it has minimal impact on the in vivo inflammatory response, or innate immunomodulatory capacity, in women selecting C‐section.


Introduction
Obesity is a global epidemic affecting more than 1.9 billion adults [1]. Increased societal obesity is a massive burden on health care systems and society due to its direct and indirect costs [2,3]. Its impacts now exceed those of smoking as the most expensive preventable disorder [3].
Obesity during pregnancy is associated with increased meta-inflammation [4][5][6][7][8]. It is one of the most common obstetrical risk factors for adverse outcomes [9]. It is linked to increased risk of gestational diabetes, placental inflammation, preeclampsia, miscarriage, preterm birth, caesarian section, and stillbirth [4,6,7,10,11]. The clinical complications arising from maternal obesity are doseresponsive and associate directly with increasing BMI [6]. Thus, preeclampsia, the leading cause of maternal and perinatal morbidity, exhibits a stepwise correlation with increasing BMI class [10].
The impact of obesity in pregnancy is not limited to maternal health. Increasing evidence indicates influences of maternal physiology on outcomes in their children that range from development of a healthy microbiome [12] to increased risks of metabolic dysfunction, heart disease, and other chronic inflammatory conditions [13,14]. Taken together, the increasing frequency and intensity of obesity in women of child bearing age is of great concern [9,15].
Surgery results in multiple intra-and post-operative alterations in all individuals. These include innate immune activation and rapid systemic increases in circulating inflammatory biomarkers that result from the combined impact of tissue damage, anesthesia, postoperative pain, and psychological stress [16][17][18]. In some individuals, surgery leads to intense systemic inflammatory response syndrome and can result in multi-organ failure [17]. The specific contributions of obesity to normal inflammatory processes, as well its impact on subsequent clinical outcomes including infection, wound healing, embolism, and mortality are areas of active investigation [19].
Whether Caesarian section elicits more pronounced inflammatory responses in obese women, and/or any counterbalancing changes in anti-inflammatory responses, is unknown. Given that obesity increases the likelihood of women receiving planned or emergent C-sections [6], it is important to better understand the impact of surgery on pregnant women. Here we test the supposition that pregnant women of BMI 35-40 at term develop exaggerated net inflammatory responses to C-section. Healthy, termpregnant, obese women were compared with non-obese, term-pregnant controls to examine in vivo and in vitro innate immune capacity. In vivo responses were examined as changes in systemic induction of pro and anti-inflammatory biomarkers and acute phase proteins prior to and during the acute surgically induced (C-section) innate response. Independently, innate immune capacity following surgery was assessed by acutely stimulating fresh PBMC, obtained 24 h after surgery, with a panel of defined PRR-ligands and comparing pro-and anti-inflammatory cytokine responses. The results demonstrate that while surgery elicits intense acute responses in vivo, their magnitude is indistinguishable in the two populations of women, despite the fact that the obese women exhibit higher baseline inflammatory marker expression pre-C-section. Similarly, in vitro stimulation of innate cytokine responses revealed no differences. The results argue that while obesity is clearly to be minimized or avoided, the acute systemic innate immune response to C-section surgery, or to subsequent stimulation with microbial mimics, is not detectably different in Class II obese women.

Participants
Following ethics approval from University of Manitoba Institutional Review Board and written informed consent from each participant, 36 healthy pregnant women were enrolled within a day of elective caesarean section (Fig. 1). Sixteen were Class II obese [20] (mean BMI at recruitment 37.7 AE 2.7), and 22 were healthy pregnant controls (mean BMI 28.4 AE 1.7). Table 1 provides key clinical characteristics of the participants. Inclusion criteria included: Uneventful term pregnancy without documented concerns about hypertension, proteinuria, or gestational diabetes resulting in a healthy baby. One woman had twins. Exclusion criteria included labor at time of C-section, comorbid inflammatory disease, other chronic diseases (e.g., osteoarthritis or asthma) or current infection. All denied any recent illness. Heparinized blood was drawn at IV start, then again 1, 3, 5, and 24 h post-surgery in the recovery room. Plasma was collected by centrifugation (500g, 10 min), aliquoted and stored at À208C.

In vitro culture
Innate stimuli (PRR ligands) representative of bacterial and viral stimuli were used for short term in vitro stimulation of fresh PBMC directly ex vivo. Briefly, PBMC obtained 24 h post-partum were isolated using Ficoll (GE Healthcare, Mississauga, Canada) gradients, cultured in triplicate at 350,000 cells/well with 200 mL medium alone and in the presence of a TLR4 ligand (0.4 ng/mL LPS, InvivoGen, San Diego, CA), TLR8 ligand (400 ng/mL CL075, InvivoGen), TLR3 ligand (50 mg/mL poly(I:C), InvivoGen) or RLR ligand (100 ng/mL poly(I:C)/Lyovec, InvivoGen). Supernatants were harvested after 24 h culture and stored at À208C.

Immunological analyses
Pre-and post-operative samples for individuals were assayed as pairs to control for inter-assay variation. Meso Scale Discovery (MSD, Rockville, MD) singleplex assays were used to quantify CRP, CCL2, CCL8, CXCL10, TNFa, IL-6, IL-10, IL-1Ra, and sTNF-RII following manufacturer's protocols. Leptin and PTX3 (R&D Systems, Minneapolis, MN) were quantified with ultrasensitive ELISA protocols as described [21,22]. The concentrations reported are based on standard curves created using serial dilutions of fresh aliquots of constant recombinant lab standards in medium stored at À808C in individual 400 mL aliquots (Cedarlane, Burlington, Canada; PeproTech, Quebec, Canada; R&D Systems). The median assay coefficient of variation was typically below 5% for MSD assays and between 5% and 15% for ELISA.

Statistics
Each point represents the average value obtained from duplicate or triplicate analyses of an individual woman's plasma or culture supernatant at that time. Intragroup data were analyzed using pairwise comparisons of medians (Wilcoxon Matched Pairs/Signed Rank tests, GraphPad Prism, La Jolla, CA). Comparisons of obese versus control populations utilized Mann-Whitney analysis. Differences were considered significant at the 95% confidence level (twotailed p < 0.05).

Systemic innate immune status in vivo prior to C-section
In this longitudinal study, two populations of healthy pregnant women who gave birth to healthy full term infants by elective C-section after uneventful pregnancies were studied. Initially, to determine the extent to which pregnant women if BMI 35-40 at term exhibited baseline elevations in inflammatory biomarkers, plasma samples were assessed for leptin and other biomarkers characteristic of meta-inflammation. Figure 2 demonstrates that pregnant women of BMI 35-40 have higher baseline leptin and CRP levels at term than do healthy pregnant controls (p ¼ 0.0002, 0.001, respectively). Pentraxin-3, an early biomarker of acute inflammatory response [23] was indistinguishable between groups (p ¼ 0.70). Among systemic innate cytokines and chemokines, median CCL2 was $40% elevated in obese women prior to surgery, whereas plasma TNFa was indistinguishable.
Kinetics of biomarker expression following elective C-section Different innate stimuli (i.e., surgery, trauma, infection, immunization) elicit very different kinetics of expression for innate immune cytokine responses. To define (1) which biomarkers were increased upon surgery; and (2) the optimal time points to examine such responses, five pre-/ post-operative time points were examined. Kinetics of proand anti-inflammatory responses following C-section are shown in Figure 3. For those biomarkers that did demonstrate responses, increases were substantial, ranging from 3 to 13-fold. Several other biomarkers that were readily found in plasma pre-surgery, did not exhibit increased expression following C-section (i.e., PTX3, TNFa, IL-10, Fig. 3) as well as IL-6 and sTNF-RII (data not shown). Inflammatory responses to C-section in normal and Class II obese women C. Graham et al.

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Pro-inflammatory, anti-inflammatory, and acute phase protein responses in obese women post-surgery Two hypotheses were tested. Given the chronic mild inflammatory status characteristic of obesity, we hypothesized that obese women undergoing C-section would exhibit more rapid kinetics of response. However, no differences were evident in the kinetics of pro-inflammatory, antiinflammatory, or acute phase protein responses (Fig. 4). Note that the specific times shown for each cytokine differ slightly so as to capture the development and peak expression for each. Secondly, the hypothesis that pregnant obese women exhibit more intense innate responses to C-section surgery than do non-obese women was tested. Again, systemic responses to surgery for panels of pro-inflammatory (Fig. 3A), anti-inflammatory (Fig. 3B), and acute phase proteins (Fig. 3C) were intense yet indistinguishable for each of the biomarkers examined. Thus, neither the kinetics nor intensity of pro/anti inflammatory responses was altered in pregnant women with Class II obesity.

Innate immune activation to acute PRR-selective activation in vitro
To determine if acute innate immune activation with different stimuli (e.g., PRR-ligands vs. surgical trauma) elicits differential responses post-operatively, fresh PBMC  Inflammatory responses to C-section in normal and Class II obese women C. Graham et al.

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were obtained 24 h post-C-section and stimulated in vitro with ligands characteristic of bacterial and viral stimuli (Fig. 5). Fresh PBMC were used, rather than purified monocytes, for two reasons. Firstly, an extensive range of circulating cell types (the frequency of each of which varies widely between different individuals) produce cytokines and chemokines upon PRR-stimulation. We wished to sample the broader repertoire of circulating cells capable of responding to PRR ligation, thereby providing a global view of changes among obese women. Secondly, less intellectually important but logistically relevant, the limited blood volumes available from pregnant women would have reduced the n of women available for the study as well as the number of analyses that could be performed with isolated monocytes. As anticipated, TLR4/LPS stimulation elicited strong pro-and anti-inflammatory responses. The intensity of each response was indistinguishable when comparing normal and obese populations. Similarly, among viral surrogates, TLR8 (CL075) and TLR3 (poly(I:C)), stimulation yielded strong pro-and anti-inflammatory cytokine production in vitro. Again, the two populations of women responded indistinguishably. Finally, examination of a RLR-stimulus also elicited strongand indistinguishable-responses in the two populations. Thus, a broad panel of pro-and anti-inflammatory responses evoked by different microbial mimics were vigorous, yet very similar in both populations following elective C-section.

Discussion
Obesity is associated with a plethora of undesirable consequences. Despite a wide variety of public health initiatives, it is has been extremely resistant to change [24]. Given that C-section is increasingly common in the developed world, and that obese women are more likely to require C-section birth [6], improved understanding of complications resulting from such surgery is needed. Here we demonstrate that while pregnant class II obese women (BMI 35-40) exhibit constitutively elevated expression of inflammatory biomarkers (meta-inflammation) at full term pregnancy, their in vivo and in vitro innate responses to surgery or to innate immune (PRR-dependent) stimulation are essentially indistinguishable from those of healthy controls. There is no question that obesity is an undesirable state associated with a multitude of health risks for both mother and, in subsequent years, offspring. However, it is important to assess what biological processes and outcomes are demonstrably affected by elevated BMI and which are not. The data in this report suggest that C-section in itself does not create a differential impact on a panel of 11 in vivo pro-, anti-inflammatory, and acute phase protein responses beyond that normally induced by C-section itself. Complementing this perhaps surprising conclusion, a related study of inflammation in pregnant rodents fed high fat ''cafeteria diets'' reached similar conclusions [25]. A complementary study, focussed on putatively differential development of inflammation in vivo in lean and obese Inflammatory responses to C-section in normal and Class II obese women C. Graham et al.

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women over the course of pregnancy (i.e., from recruitment up to delivery), concluded that there was no evidence of additive or synergistic effects between adiposity and pregnancy in terms of endogenous maternal levels of proinflammatory markers during gestation [4]. While these findings appear paradoxical, there is an increasing literature indicating that in some circumstances obesity (<Class II) do not harm and, in some specific circumstances, can be beneficial. For example, low levels of obesity are associated with improved survival during septic shock, with the obese patients exhibiting the lowest 28-day mortality while lean patients (BMI <25 kg/m 2 ) experienced the highest mortality (p ¼ 0.02) [26].
This study has caveats that are important for its interpretation. BMI is a widely used but imperfect measure of obesity [19,27]. It does not distinguish lean tissue mass from adipose tissue mass and does not reflect adipose tissue distribution. Measures such as visceral adiposity may provide more specific tools for investigating relationships between obesity and biological outcomes. However, such measures were not available to us for populations of term pregnant women. BMI continues to be widely used today because it is readily determined and is considered by many to be the most useful tool available from a broad-based health policy perspective [28]. Secondly, this study focused on obesity at term and its associated risks for women of elevated BMI (35-40) who elect C-section surgery. The goal was not to address relative levels of weight gain or their impact on maternal physiology over the term of gestation. Finally, we certainly recognize that of the large number of immuneregulatory cytokines known, there may be other immune modulators that do exhibit a differential response to Csection delivery in obese women. Nevertheless, among the 11 pro-inflammatory, anti-inflammatory, and acute phase proteins examined, there was no evidence of differential responses in vivo or in vitro.
Several avenues of future research are suggested by our findings. The relative proportion of pro-and antiinflammatory molecules produced by adipose tissue versus innate immune cells is not known. Because the focus was on the systemic impact of an acute innate immune stimulus (here, the surgical impact of C-section delivery), net plasma biomarker expression was examined, independent of source. Potential differences over the weeks and months postdelivery were not examined, nor were inflammation levels in offspring. Similarly, work will be needed to define postoperative complications and clinical status during that period and its relationship to inflammatory responses. Certainly among morbidly obese mothers (BMI > 50), a population less commonly represented in society or successfully pregnant women, the incidence of clinical complications is substantially enhanced in the days and weeks post-delivery [29].
Finally, interpretation of these findings requires attention to the recruitment criteria utilized here. To test the hypothesis that obesity, in itself, is linked to enhanced inflammatory responses to surgical trauma in pregnant women, participants with hypertension, proteinuria, diabetes, or other ongoing chronic inflammatory diseasesconditions over-represented in obese populations, were excluded. All participants were healthy, pregnant women. The impact of C-section on women with extensive comorbidities was beyond the scope of this study and will require much larger populations and stratification by the different constellations of comorbidities that are overrepresented in obese women.

Conclusion
C-section, like any surgery, elicits systemic responses by multiple pro-and anti-inflammatory constituents. Here we demonstrate that neither the kinetics nor the maximal intensity of a broad panel indicators of innate activation differed when comparing healthy normal and Class II obese women's responses to caesarian section. Similarly, acute stimulation of PBMC ex vivo with a panel of PRR ligands representing mimics of bacterial or viral infection elicited indistinguishable pro-and anti-inflammatory responses in vitro. The data suggest that while Class II obesity is not desirable, it has minimal impact on the in vivo inflammatory response, or innate immune capacity, of women undergoing C-section.