A Novel Model of House Dust Mite-Induced Maternal Allergic Asthma and Neuroinflammation in the Offspring

A Novel Model of House Dust Mite-Induced Maternal Allergic Asthma and Neuroinflammation in the Offspring | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A Novel Model of House Dust Mite-Induced Maternal Allergic Asthma and Neuroinflammation in the Offspring Hadley Osman, Juan Tamayo, Kent Pinkerton, Paul Ashwood This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8745297/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 13 You are reading this latest preprint version Abstract Maternal immune activation (MIA) through infection, toxic exposure, or chronic inflammatory disease has been linked with an increased risk of neurodevelopmental disorders, in offspring, such as autism spectrum disorder (ASD) or schizophrenia. Previously we have shown that inducing maternal allergic asthma (MAA) with ovalbumin increased pro-inflammatory cytokines during pregnancy and altered behaviors in offspring. Other studies have separately shown that particulate matter (PM) can exacerbate the effects of allergic asthma. In this study, we used house dust mite (HDM) to induce MAA combined with a pre-pregnancy exposure to PM to assess the possible combinatorial effects on maternal inflammation and offspring brain cytokine levels. BALB/c mice were treated preconception with PBS, HDM, PM, or HDM + PM, and subsequently challenged with HDM or PBS on gestational days (GD) 12.5 and 15.5. Maternal serum, placental tissue, and fetal brains were collected on GD15.5. Offspring brains were collected at postnatal day 15 to analyze for differences in cytokine levels across treatments. HDM and HDM + PM treated dams were found to have elevated levels of pro-inflammatory and T-helper (T H ) type 2 cytokines in the serum and increased inflammation in the lungs, consistent with an allergic response. In the placenta, increased T H 2 cytokines IL-4 and IL-5 were found in the HDM + PM offspring, consistent with the maternal serum data. Fetal brains had a decrease in MIP-2, MCP-1, and G-CSF in HDM and HDM + PM treatment groups. In the p15 hippocampus, HDM and HDM + PM groups showed increases in several pro-inflammatory cytokines but decreased regulatory cytokine IL-10, representing a skewed neuroinflammatory immune profile. We also saw decreased microglia density in the HDM + PM group in the hippocampus. Altered cytokine profiles were additionally noted in the cortex and cerebellum of p15 offspring. Overall, this data shows an increased pro-inflammatory response and a decreased regulatory response in the brains of offspring in response to maternal immune activation with HDM and PM. Autism Maternal Immune activation (MIA) Maternal allergic asthma (MAA) Cytokines regulation microglia Lung Brain Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder that has increased in prevalence, with 1 in 31 children in the US being diagnosed by the age of 8 (Shaw et al., 2025). While the underlying etiology of ASD is unknown, several genetic and environmental factors have been associated with an increased risk of ASD (Goines & Ashwood, 2013 ; Sealey et al., 2016 ). During fetal neurodevelopment, maternal inflammation can have lasting impacts on offspring brain function that can lead to neuroinflammation and behavioral alterations. For instance, maternal infections that resulted in hospitalization during early pregnancy were significantly associated with birthing a child later diagnosed with ASD (Atladottir et al., 2010). Animal models have further demonstrated this effect by using viral and bacterial mimics to assess altered neurodevelopment, behavior, and neuroinflammation in the offspring (Choi et al., 2016 ; Haddad et al., 2020 ; Schwartzer et al., 2013 ; Simões et al., 2018). In addition, chronic inflammatory conditions during pregnancy are also linked with an increased risk of ASD (Croen et al., 2005 ). Maternal asthma has been identified as the most common maternal immune condition, to increase risk for ASD in population-based studies (Patel et al., 2020 ; Croen et al., 2024; Croen et al., 2019 ). Asthma is a T-helper type 2 (T H 2) driven chronic inflammatory condition typically characterized by an increase in IL-4, IL-5, and IL-13, along with immune cell infiltration of the airways (Gans & Gavrilova, 2019). During pregnancy, asthma symptoms can become exacerbated and lead to an increase in hospital visits and hospitalization (Abdullah et al., 2020 ). Furthermore, animal models of ovalbumin (OVA) induced maternal allergic asthma (MAA) have shown an increase in marble burying behavior and a decrease in social interactions in offspring of exposed dams (Schwartzer et al., 2013 ; Church et al., 2021 ). In the same model, offspring showed increased pro-inflammatory cytokines in the fetal whole brain (Tamayo et al., 2022 ). Increased neuroinflammation was also seen in postnatal day (p)15 and p35 hippocampus and cortical regions (Tamayo et al., 2023 ). While a useful tool for animal models of asthma, OVA is not the most physiologically relevant asthma causing agent in humans. House dust mite (HDM) is a common allergen associated with asthma in humans, and many studies are shifting towards using natural allergens, like HDM, to induce asthma in animal models (Castañeda & Pinkerton, 2016 ). However, HDM has not yet been used in mouse models to study MAA and effects on offspring neurodevelopment. Particulate matter (PM) exposure has been shown to exacerbate asthma symptoms caused by HDM (Castañeda & Pinkerton, 2016 ). In the environment, PM can vary greatly in composition, while exposed individuals can vary in terms of the time of onset and duration of exposure (Castañeda & Pinkerton, 2016 ; Carosino et al., 2015 ). Even if only introduced during the sensitization phase of asthma, PM can exacerbate the hallmarks of asthma such as cellular infiltration, airway inflammation, and oxidative stress (Carosino et al., 2015 ). PM exposure has also been linked to an increased risk of ASD (Talbott et al., 2015 ). Due to these associations, this study aimed to investigate whether PM exacerbation of MAA during the sensitization period led to altered neuroinflammatory profiles in the offspring. We used a novel experimental model of HDM-induced MAA and pre-pregnancy exposure to PM collected from the central valley in California to determine the effect of each exposure, separately and in combination compared with PBS controls, on maternal responses during pregnancy and the offspring cytokine profiles in the brain. We hypothesized that exposure to PM during the sensitization phase would exacerbate maternal asthma and lead to increased inflammation in offspring brains in HDM + PM groups. Methods Animals BALB/c mice from Envigo Laboratories (Livermore, CA) were housed with same sex littermates at the Center for Health and the Environment at University of California, Davis in Davis, CA. Mice were kept in a 23 o C room with a 12-hour light/dark cycle and food and water provided as needed. Procedures and protocols were performed with approval by the University of California, Davis Institutional Animal Care and Use Committee and following the National Institute of Health Guide for the Care and Use of Laboratory Animals. Maternal allergic asthma and particulate matter exposure paradigm All mice were purchased at 7–8 weeks of age. After allowing mice to adjust to their environment for one week after purchasing, female mice were anesthetized with isoflurane and sensitized intranasally with 25 µl of 1 mg/mL HDM resuspended in phosphate buffered saline (PBS) three times in one week, every other day. Mice in the PM and HDM + PM groups were also exposed intranasally to 33.3 µl of 1 mg/mL PM. Mice were then paired for mating with two females per one male and checked daily for the presence of a seminal plug. Presence of a seminal plug was noted as gestational day (GD) 0.5. On gestational days 12.5 and 15.5, asthma was induced in pregnant females by intranasally exposing dams to 25 µg of HDM, in the HDM and HDM + PM groups. The PBS control group or PM alone group were given intranasal PBS during the asthma induction phase. Tissue collection and lysis Four hours following the final exposure on GD 15.5, a subset of dams from each group were euthanized using CO 2 for collection of maternal serum and lung tissue. The remaining dams were left undisturbed to give birth naturally. On postnatal (p) day 15, offspring were anesthetized with isoflurane and underwent a terminal cardiac perfusion with ice cold PBS. Following perfusion, brains were collected and micro dissected to separate the cortex, cerebellum and hippocampus. Brain tissue was flash frozen and stored at -80 o C for future use. Tissue was lysed using cell lysis buffer (Cell Signaling, Danvers MA) and protein from the tissue lysate was quantified using a Bradford assay. Lung collection and analysis At necropsy, the trachea of the maternal mice was cannulated, the right mainstem bronchus ligated, and right lobes removed, while the left lung was inflation-fixed with 4% paraformaldehyde at a hydrostatic pressure of 30 cm for one hour. The left lung was placed for 24 hours in fixative prior to transfer into 70% ethanol for storage. The fixed left lung was subsequently cut into four transverse sections, dehydrated in a graded series of ethanol and xylene, and embedded in paraffin. A rotary microtome was used to prepare 5 µm thick tissue sections stained with hematoxylin and eosin for viewing. Lung tissue sections were evaluated using a semi-quantitative grading system for inflammation of the airways, blood vessels, alveoli, and pleura. This scoring system evaluated the extent (0 to 3) and severity (0 to 3) of inflammation for each region of the lungs. Cytokine and Chemokine analysis Cytokines and chemokines in maternal serum and offspring tissue lysates were measured using a magnetic bead 25-plex immunoassay (Milliplex Mouse Cytokine/Chemokine Magnetic Bead Panel #MCYTMAG70PMX25BK). The following cytokines, chemokines, and growth factors were analyzed: granulocyte colony stimulating factor (G-CSF), granulocyte colony stimulating factor (GM-CSF), IFN-γ, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17, interferon gamma-induced protein 10 (IP-10), keratinocyte chemoattractant (KC), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1 alpha (MIP-1α), MIP-1β, MIP-2, chemokine ligand 5 (CCL5/RANTES), and tumor necrosis factor alpha (TNF-α). The plate was washed prior to adding standards and samples following the manufacturer’s protocol. For maternal serum, 25 µl of serum was loaded onto the plate. For brain tissue lysates, protein concentration was normalized so that 100 µg of protein was added to each well in duplicate, along with appropriate standards and quality controls. Antibody-coupled magnetic beads were added to each well and incubated overnight. After incubation, the plate was washed following the manufacturer’s protocol and then incubated with biotinylated detection antibody, and then with streptavidin-phycoerythrin. The plates were read on a Bio-Rad Bio-plex 200 plate reader (Bio-Rad Laboratories, Hercules, CA). The following are the minimum levels of detection for each analyte: G-CSF: 1.7 pg/mL; GM-CSF: 10.9 pg/mL; IFNγ: 1.1 pg/mL; IL-1α: 10.3 pg/mL; IL-1β: 5.4 pg/mL; IL-2: 1.0 pg/mL; IL-4: 0.4 pg/mL; IL-5: 1.0 pg/mL; IL-6: 1.1 pg/mL; IL-7: 1.4 pg/mL; IL-9: 17.3 pg/mL; IL-10: 2.0 pg/mL; IL-12 (p40): 3.9 pg/mL; IL-12 (p70): 4.8 pg/mL; IL-13: 7.8 pg/mL; IL-15: 7.4 pg/mL; IL-17: 0.5 pg/mL; IP-10: 0.8 pg/mL; KC: 2.3 pg/mL; MCP-1: 6.7 pg/mL; MIP-1α: 7.7 pg/mL; MIP-1β: 11.9 pg/mL; MIP-2: 30.6 pg/mL; RANTES: 2.7 pg/mL; TNF-α: 2.3 pg/mL. If more than 60% of samples fell out of range they were excluded from analysis. For cytokines with only a few samples out of range, the sample was given a proxy value of one half the minimum level of detection for that analyte. Microglia density analysis For analysis of microglial density, in the hippocampus corresponding to region 72 in the Allen Brain Atlas, we closely followed a protocol used previously in our studies (Tamayo et al., 2023 ). Briefly, after perfusion, right hemispheres were fixed in PFA, then transferred to a sucrose gradient. They were sliced using a cryostat into 20um sections. The 6th well of sections was used for IBA1 staining using DAB substrate. The sections were imaged at 20x on a brightfield microscope (Nikon Eclipse Ci, Nikon, Tokyo) and merged using imageJ. Merged images were then analyzed using an imageJ script outlined in Marques et al., 2023. Microglia were also manually scored by an unbiased and blinded laboratory member to confirm accuracy of our semi-automated scoring. Statistical analysis All statistical analysis was performed using GraphPad Prism v10.2.0 (GraphPad Software, Boston, MA). Outliers Identity was performed via ROUT and testing was performed on data excluding any outliers. One-way ANOVA with multiple comparisons and Sidak adjustment for multiple comparisons was run to determine statistical significance with p < 0.05 determining significance. Descriptive statistics including mean, median, and interquartile range were also obtained. Heat maps were made using the p-values obtained from the ANOVA. We also ran principal component analysis (PCA), selecting principal components based on parallel analysis with a percentile level of 95%. Symbol fill color was determined by the categorical variable, which was treatment group. Cytokines were used as continuous variables. For correlation analyses, data was separated by treatment group and r was computed for every pair of Y data sets, creating a correlation matrix. This data shows how patterns of cytokines vary in each sample in relation to other cytokines, and reveals valuable information that may not show up on statistical analyses of cytokine levels alone. Microglia counts were analyzed using one-way ANOVA with adjustment for multiple comparisons. Results Maternal lung inflammation Semi-quantitative analysis of the maternal lungs showed no significant differences between the PBS control mice or the PM-sensitized mice in the airways, blood vessels, alveoli, or pleura. In contrast, significant differences were found in the degree of inflammation for all four regions in the HDM and HDM + PM exposure groups. One notable finding was a statistically significant increase in the degree of alveolar inflammation in the HDM + PM group in comparison to the HDM-only group ( p = 0.0335). Maternal Serum Cytokine Analysis Serum cytokines were measured at GD15.5 in dams to determine systemic inflammatory responses in each treatment group. A significant increase in T H 2 cytokines was seen in the maternal serum in HDM and HDM + PM groups compared to PBS controls (Fig. 1 a and 1 b). In HDM and HDM + PM dams, IL-5 (HDM p = 0.0345; HDM + PM p < 0.0001) and IP-10 (HDM p < 0.0001; HDM + PM p = 0.0002) were increased, with IL-5 more significantly increased in the combined exposure group than HDM alone (Fig. 1 a and 1 b). IL-4 was increased in the HDM dams ( p = 0.0049) and trended towards increase in the HDM + PM dams ( p = 0.0550) compared to PBS alone but did not reach significance after corrections for multiple comparisons. The PM alone group did not have an inflammatory effect on maternal cytokine responses when given antenatally in this model. Overall, combined with data from lung tissue, this shows that our model of HDM allergic asthma induced a T H 2 skewed, inflammation, and asthma-like condition in the dams. On PC analysis, several data points from the HDM + PM group clustered away from the rest of the data, but no other apparent separations were seen. Correlation analysis shows a shift towards strong negative correlations in the PM group. There was increased strength of positive correlations between cytokines like IL-13, IL-15, and IL-17 in the HDM + PM group (Fig. 1 d). Overall, correlation analysis shows us several patterns between cytokines that were otherwise not significant, especially the changes to the PM group, as none were seen in mean differences in Fig. 1 a. GD15.5 placental cytokine profiles In the placenta, an overall increased T H 2 cytokine profile was seen, with IL-5 increased in both HDM ( p = 0.0326) and HDM + PM ( p = 0.0053) offspring and IL-4 increased in only the HDM + PM offspring (Fig. 2 a and 2 b). Increased KC in the HDM + PM offspring compared to the PBS controls was also observed (Fig. 2 a and 2 b). After performing PC analysis, HDM + PM data separated from PBS and HDM along PC2 (Fig. 2 c). The PC1 accounted for 40.1% of variance between data and PC2 accounted for 19.92% of variance between data. Furthermore, comparing cytokines using a correlation matrix, several important differences were noted between the treatment groups. PBS placentas were on the whole positively correlated, with no strong correlation between T H 2 cytokines. The strongest correlation in the PBS group was seen between IL-1α and TNFα. In the HDM and HDM + PM groups, positive correlations were seen between the T H 2 cytokines IL-4 and IL-5, which were both significantly elevated in the placenta (Fig. 2 d). In HDM + PM, a strong correlation between these T H 2 cytokines and IL-6 was also observed (Fig. 2 d). When analyzed separately, male and female offspring did not have significant differences (data not shown). GD15.5 fetal brain cytokine profiles In the fetal brain, decreased G-CSF and MCP-1 was observed in the HDM (G-CSF p = 0.0001; MCP-1 p = 0.0472) and HDM + PM offspring (G-CSF p < 0.0001; MCP-1 p = 0.0023). In addition, HDM + PM offspring had decreased MIP-2 ( p = 0.0119) (Fig. 3 a and 3 b) PCA showed a clear separation of PM from the other treatment groups along PC2 with PC1 accounted for 42.21% and PC2 accounts for 17.77% of the variance of the data (Fig. 3 c). Correlation matrices showed several differences between treatment groups. In the PBS offspring, IL-1α was positively correlated with MCP-1, IL-9, and IP-10; and IL-10 was positively correlated with MIP-2 (Fig. 3 d). In contrast, PM offspring showed a negative correlation between IL-1β and MIP-1α. In HDM, IL-1α was correlated with IL-1β, IL-2, IL-10, and MIP-1α. The HDM + PM offspring had strong positive correlations between G-CSF and MCP-1, IL-2, IL-1α, and IL-9; in addition, IL-9 correlated with IL-10 and IL-1β; and, the chemokine MCP-1 correlated with IP-10 (Fig. 3 d). Taken together these correlations showed a distinct pattern of cytokines in the fetal brain depending on the mother’s treatment group. There were no significant differences between male and female fetal brain cytokines (data not shown). p15 Cortex cytokine profiles In the cortex, PM alone showed a significant decrease in IFNg ( p = 0.0120) compared to controls. In HDM alone, there were no significant changes when compared to controls. In the combined HDM + PM exposure group, significantly decreased IFNg was found, and there was also increased MIP-2 ( p = 0.0077), compared to both PBS controls and the HDM alone group. IL-17 trended towards increased levels in HDM + PM ( p = 0.0501). The cortical cytokine data suggested a combined effect of HDM and PM on the offspring brain. There was no obvious separation between groups upon PC analysis of PC1 and PC2. PC1 accounted for 30.93% of variance in data and PC2 accounted for 19.52% (Fig. 4 c). When investigating correlations between significant cytokines, no strong correlations were observed with IFNg in any treatment groups. MIP-2 was correlated with GM-CSF in PBS, but was not strongly correlated with any cytokines in the other treatment groups. IL-17 was positively correlated with IL-13 in PBS controls, and with IL-6 in PM, and with IP-10 in HDM + PM. Interestingly, IL-6 and KC were positively correlated in both PBS and HDM + PM offspring (Fig. 4 d). p15 Cerebellum cytokine profiles In the cerebellum there were no significant changes seen in cytokine levels in HDM alone or PM alone groups compared to PBS controls in the p15 offspring. However, significantly decreased levels of IL-4 (p = 0.0424) and G-CSF (p = 0.0048) were observed in the HDM + PM group when compared to PBS controls (Fig. 5 a and 5 b). There was no separation between groups along the PC2 axis, with only a slight skewing of the HDM + PM data points along the PC1 axis (Fig. 5 c). IL-4, which was significantly decreased in the cerebellum of p15 offspring, was positively correlated with IL-2 and IL-1α. IL-4 was also correlated with IL-2 in both PM and HDM + PM group, but was not significantly associated in HDM. In the HDM + PM offspring, IL-4 was positively correlated with IL-6 and IL-15 (Fig. 5 d). The data showed an overall decrease in regulation and growth factor production in the cerebellum in the dual exposure group, but with no major impact of either of the single exposure groups. p15 Hippocampus cytokine profiles In the hippocampus of both HDM and HDM + PM offspring, significantly increased IL-6 (HDM p = 0.0004; HDM + PM p = 0.0001), IL-9 (HDM p = 0.0005; HDM + PM p = 0.0206), and TNFα (HDM p = 0.0001; HDM + PM p = 0.0001) was observed (Figs. 6 a and 6 b). In contrast, IL-10 (HDM p = 0.0001; HDM + PM p = 0.0001), IP-10 (HDM p = 0.0187; HDM + PM p = 0.0079), IL-1α (HDM p = 0.0053; HDM + PM p = 0.0027), and IL-1β (HDM p = 0.0027; HDM + PM p = 0.0006) were significantly decreased in both HDM and HDM + PM groups compared to the PBS group (Fig. 6 a). KC levels were decreased only in HDM + PM ( p = 0.0164) compared to PBS controls (Fig. 6 a). These data show significantly altered inflammatory and anti-inflammatory cytokine profiles in both HDM and HDM + PM offspring, with a larger effect seen in the combined exposure group for some cytokines. PC analysis shows clear separation of HDM and HDM + PM groups from PBS and PM alone groups along the PC1 axis. Some separation between HDM and HDM + PM also occurred along the PC2 axis. PC1 in the hippocampus was responsible for 31.26% of variance in the data and PC2 was responsible for 29.8% (Fig. 6 c). In the PBS group, cytokines seen as significant in Fig. 4 a and 4 b were positively correlated with each other. However, when examining the PM and HDM data, these positive correlations were almost completely absent. In the HDM + PM group, no cytokines were significantly correlated with each other, positively or negatively (Fig. 6 e). In addition, no sex differences in cytokines were observed after adjustments for multiple comparisons. p15 Hippocampal microglia density As we saw the most alterations in the hippocampal cytokines, we chose to do additional analysis on the microglial density. Moreover, prior work in our OVA models showed changes in microglia only in the hippocampus at p15. Allen brain atlas section 72 was used as reference to choose sections for analysis. After using a semi- automatic method for cell counting and manual counting by an unbiased and blinded laboratory member, the density of microglia was determined. In this region of the hippocampus significantly decreased microglia density was observed in the HDM + PM group when compared to the PBS group ( p = 0.0420) (Fig. 6 d). PM and HDM alone did not yield any significant differences when compared to PBS controls. Discussion Maternal asthma and exposure to PM exposure have both been linked with an increased risk of neuroinflammation in the offspring (Croen et al., 2005 ; Tamayo et al., 2023 ; Talbott et al., 2015 ). Previous studies have used an OVA-based regimen to model MAA, while in the current study the more physiologically relevant allergen HDM was used to induce an asthma response in the dams. Given that PM can aggravate asthma during sensitization, we hypothesized that the addition of PM would exacerbate maternal systemic inflammation in the dams and increase neuroinflammatory responses in the offspring brain. We saw an additive effect of PM on maternal asthma induction with elevated T H 2 cytokine in the maternal serum and in the alveolar region of the lungs. In addition, when analyzing brain samples taken from p15 offspring there was an increased additive effect on cytokine levels when dams were exposed to both PM and HDM. Allergic asthma is associated with a T H 2 immune response. Asthma is a highly heterogeneous disease, with subtypes of eosinophilic (T H 2 driven), neutrophilic (T H 17 driven), or combined (eosinophilic + neutrophilic) (Gans & Gavrilova, 2019). Maternal serum data confirmed a systemic maternal inflammatory response in the HDM and HDM + PM groups. The cytokines seen in maternal serum suggested a T H 2 response in HDM exposure groups due to increased IL-4, IL-5, and IL-6. The cytokines IL-4, IL-5, and IL-13 are commonly elevated in asthma patients (Lambrecht et al., 2019 ) with IL-6 also seen in T H 2 immune reactions (Wong et al., 2001 ). Furthermore, we saw increased lung inflammation in both HDM and HDM + PM groups, consistent with allergic response. Interestingly, both maternal sera and lung data showed an additive increased inflammatory response in the HDM + PM group over the HDM alone, suggesting PM primed a more exaggerated maternal response. The placenta is the primary source of communication between the mother and developing fetus. Cytokines can be produced by placental cells, especially placental macrophages, and can alter the placental microenvironment, potentially leading to changes in fetal development (Ben-Rafael & Orvieto et al., 1992). During the entire pregnancy, but increasingly so in the third trimester in humans, cytokine profiles in the placenta skew towards T H 2, which helps prevent rejection of the fetus (Dealtry et al., 2000 ; Doria et al., 2006 ). However, pregnancy complications have also been noted to occur with dominant T H 2 responses. In this model, we saw a significant increase in the T H 2 cytokines such as IL-4 and IL-5 in the placenta of HDM and HDM + PM offspring. This mimics the pattern seen in maternal serum. Further research on isolated placental cells including macrophages which are major producers of cytokines in the placenta and participate in not only immunity but regulation of other cells in the placenta and placental morphology and formation (Pavlov et al., 2020 ) is warranted. Identifying signaling pathways associated with PM and HDM exposures may also reveal how changes in cytokine profiles can impact the fetal microenvironment and help connect the maternal, placental and fetal brain data. Cytokines are an important part of normal brain development during gestation and post-natally. Cytokines are crucial to neurodevelopment at homeostasis, and deviations from these levels, especially during key neurodevelopmental windows, have been associated with disrupted neurodevelopment (Ratnayake et al., 2013 ; Dziegielewska et al., 2000 ). In the fetal brain data decreases in 3 cytokines/chemokines were observed in the dual exposure group: MIP-2, MCP-1, and G-CSF. The chemokine MIP-2, is a neutrophil chemoattractant (Driscoll, 2000 ) and has been associated with traumatic brain injury or hypoxic conditions. However, little research has been done on whether MIP-2 can play a role in development during early neurodevelopmental windows. Altered MCP-1 levels have been noted in several human and rodent studies linked with ASD or schizophrenia, with increased levels observed in ASD individuals (Maldonado-Ruiz et al., 2022 ; Zerbo et al., 2014; Frydecka et al., 2018 ). This is in contrast to the fetal brain data form this model and may reflect dynamic changes over time and development (Robertson et al., 1994 ). The innate cytokine, G-CSF, been seen in other animal models with relevance to ASD and higher levels improved symptoms of ASD in rodent studies (Durankus et al., 2022). Mechanistically, G-CSF can interact with NMDA receptors to potentially ameliorate ASD associated behaviors (Mishra et al., 2022 ). In the MAA model, a decrease in neuroprotective G-CSF may mean a loss in NMDA receptor modulation critical for typical development. The cortex consists of gray matter that exhibits altered function and connectivity in ASD patients (Jawabri & Sharma, 2019 ; Gandal et al., 2022 ; Kana et al., 2011 ; Tamayo et al., 2023 ). In the cortex of HDM + PM mice, two cytokines were significantly altered compared to the controls. One of the cytokines, IFNγ, was decreased in the HDM + PM group. and is involved in regulating social behavior. In a mouse model using an IFNγ knockout, IFNγ −/− mice showed social deficits and altered neural circuits (Filiano et al., 2016 ). The second differentially produced cytokine/chemokine, MIP-2, was significantly increased in the cortex of the HDM + PM group. MIP-2, together with KC, can induce astrocytes to increase expression of chemokines like RANTES, IP-10, and the cytokine IL-6 (Luo et al., 2000 ). In the cerebellum, IL-4, was significantly decreased in offspring of the HDM + PM dams. In the brain, IL-4 is typically seen as regulatory and can rescue social behaviors (Derecki et al., 2010 ). IL-4 can modulate microglia, skewing them towards an anti-inflammatory state (Zhao et al., 2015 ). During the critical p15 developmental window, when synaptic pruning activity peaks, regulatory cytokines play an important role in modulating microglial activity. A decrease in these regulatory cytokines may contribute to increased neuroinflammation and over-pruning of synapses. The cerebellum is involved in motor function and learning, but also in several emotional and cognitive processes (Schmahmann & Caplan, 2006 ). In both the cortex and cerebellum, cytokines are only significant in the HDM + PM group, with several effects exacerbated compared to both PBS and HDM groups. While many changes with just HDM exposure alone are seen, differing patterns and exacerbations of allergic asthma effects by PM are evident on the offspring brain at p15. Increased IL-6 in the brain was associated with increased excitatory synapses in the hippocampus and somatosensory cortex of mice, as well as ASD-like behaviors (Wei et al., 2012 ). In this model, IL-6 was increased in maternal serum and the p15 hippocampus in offspring after HDM or HDM + PM exposures. The hippocampus is involved in social behavior, learning, memory, and spatial reasoning in the brain (Immordino-Yang & Singh, 2013 ; Rubin et al., 2014 ; Banker et al., 2021 ). Altered shape, size, and function of the hippocampus has been seen in ASD individuals (Barnea-Goraly et al., 2014 ; Dager et al., 2007 ; Solomon et al., 2015). Furthermore, several animal models show neuroinflammation in the hippocampus that was associated with alterations in behaviors, and altered microglia density (Schwartzer et al., 2013 ; Tamayo et al., 2023 ). The hippocampus is a site of adult neurogenesis, and altered inflammatory profiles may severely impact this throughout development. In humans, IL-6 was increased levels in postmortem cerebellum specimens of ASD patients (Wei et al., 2011 ). KC was the only cytokine in the hippocampus to be increased only in the HDM + PM group compared to HDM alone, or PM alone, or PBS controls. KC is a mouse homolog of the human protein growth-regulated oncogene, and has pro-inflammatory effects (Son et al., 2007 ). Levels of hippocampus IL-9, IL-10, and IP-10 were both significantly altered in the HDM and HDM + PM groups. IL-9, which was increased in HDM and HDM + PM groups, has been shown to be increased in PBMC from children with ASD (Ahmad et al., 2017 ) and is important in recruitment of T H 17 cells to the CNS (Zhou et al., 2011). IL-9 can also play a role in neurodevelopment by inhibiting programmed cell death of cortical neurons, a homeostatic process during development (Fontaine et al., 2008 ). IL-10, decreased in HDM and HDM + PM treatment groups, has important immunoregulatory functions that reduce inflammatory responses and regulate function of microglia and astrocytes in the brain. In the absence of IL-10, microglia and astrocytes are known to produce more pro-inflammatory cytokines and reactive oxygen/nitrogen species (Laffer et al., 2019 ; Zhang et al., 2020). Thus, low IL-10 in the HDM + PM exposed group would switch the profile of cytokine messages towards a more inflammatory profile. In the HDM + PM group decreased microglial density was observed in the hippocampus at p15, whereas there was no significant effect with HDM alone or PM alone. Fewer microglia in the hippocampus at p15, the peak of synaptic pruning in mice, could suggest this process is altered due to cytokine changes in the region. This can direct future studies, in which we can collect tissue for dendritic spine analysis, pre and post synaptic vesicle density, and interactions between the microglia and synaptic sites. In models of altered neurodevelopment, altered synaptic pruning is seen and associated with worsened behaviors (Kim et al., 2017 ). In postmortem ASD brains, increased dendritic spine density is seen, suggesting aberrant pruning (Hutsler & Zhang, 2010 ). In order to investigate the complex interactions of cytokines PCA and correlative analysis were performed. Along with the impact of significantly increased or decreased cytokines, altered patterns of cytokines may be revealed that were not clear when comparing mean cytokine levels. Altered cytokine ratios may play a significant role in cell function and inflammation. Future studies can then focus not only on the impact of single cytokines on neural cells, but how combinations of cytokines work together. The hippocampus was also the only region that showed clear separation of groups upon principal component analysis. Correlations of cytokines between groups showed a clear positive correlation between cytokines such as IL-9, IL-1α, IL-13, and IL-17. These correlations significantly diminished in both the PM and HDM + PM groups. In regions where few cytokines were seen as significantly changed, we were able to see striking differences in the HDM + PM correlations compared with controls. Changes between the HDM and HDM + PM correlations, in all regions and timepoints, reveal more differences between the groups than seen upon cytokine levels alone. Overall, the data from this novel model of lung-brain inflammation showed a combined effect of HDM and PM on IL-5 levels in the maternal serum, and in lung inflammation in the alveoli. The maternal exacerbation of asthma then had impacts in the offspring neuroinflammatory environment. In the placenta and fetal brain, an exacerbation of effects in the HDM + PM group over HDM or PM alone was observed. In the hippocampus, altered levels of seven cytokines in both HDM alone and HDM + PM was seen. The hippocampus also showed the most separation of groups in PC analysis, compared with the other regions of the p15 brain. Correlation analysis of all regions of the offspring brain showed changes in cytokine patterns in the HDM + PM group compared to controls, HDM alone, and PM alone. The current study several limitations including analysis of a specific PM composition. PM from different geographical regions may vary in the ability to exacerbate asthma and neuroinflammation in the offspring. Only a single time point of serum was taken, and as we do see a combined effect in more cytokines in the offspring brains, it may be plausible that cytokines in the HDM + PM maternal serum had been elevated previously or may increase later in pregnancy. In addition, this study does not include behavioral data. As this is a novel model, behavioral studies should be performed in the future due to MAA being linked to a number of neurodevelopmental disorders. Previously, ASD-like behaviors have been seen in the OVA-induced MAA model (Schwartzer et al., 2013 ). Conclusions This novel model of lung-brain inflammation that was PM presentation of HDM-induced MAA, caused a systemic inflammatory response in the dams which resulted in altered cytokine profiles in placenta, fetal brain, the hippocampus and cortex of p15 offspring. Maternal inflammation followed a T H 2-like pattern and offspring brains showed altered levels of cytokines that can have profound effects on neurodevelopmental. Several cytokines seen in this study have not been extensively studied in terms of how they may impact microglial function or neurotoxicity. The data showed some evidence for microglia activation in the hippocampus after HDM + PM that warrant further follow up. Future studies should focus on how these changes in cytokines impact neuronal connections and glial cell function. Abbreviations ASD Autism spectrum disorders HDM House dust mite PM Particulate matter MAA Maternal allergic asthma GD Gestational day PBS Phosphate buffered saline P Postnatal day IL Interleukin IFNγ Interferon gamma Poly(I C) :Polyinosinic:polycytidylic acid DAB 3,3’-Diaminobenzidine G-CSF Granulocyte colony stimulating factor GM-CSF Granulocyte colony stimulating factor IP-10 Interferon gamma-induced protein 10 KC Keratinocyte chemoattractant MCP-1 Monocyte chemoattractant protein-1 MIP-1α Macrophage inflammatory protein-1 alpha RANTES Chemokine ligand 5 TNF-α Tumor necrosis factor alpha T H T-helper CNS Central nervous system MIA Maternal immune activation Declarations Ethics approval and consent to participate The studies involved animal models and were reviewed and approved by University of California, Davis Institutional Animal Care and Use Committee (IACUC) and according to guidelines established by National Institute of Health Guide for the Care and Use of Laboratory Animals. Consent for publication Not applicable Funding This material is based upon work supported by National Institute of Environmental Health Sciences (R21ES035969) and (R21ES035492), HEDCO foundation, and the Brain Foundation. Acknowledgements We would like to thank Mr. Dale Uyeminami, Dr. Morgan Poindexter, and undergraduate Ryan C. Hart, along with all members of the Ashwood Laboratory and Pinkerton Laboratory for assistance in this project. Author Contribution HO. performed experiments, analyzed and interpreted data, and drafted the manuscript. J.T performed experiments. K.P. obtained exposures and implemented exposure panels and lung tissue analysis. P.A. formulated hypothesis, designed the study, edited the manuscript, and provided scientific funding. All authors read and approved of the final manuscript. Data Availability The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8745297","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":597242815,"identity":"c13d2264-d567-444a-ae8a-2c6ed1a06ed2","order_by":0,"name":"Hadley Osman","email":"","orcid":"","institution":"University of California, Davis","correspondingAuthor":false,"prefix":"","firstName":"Hadley","middleName":"","lastName":"Osman","suffix":""},{"id":597242819,"identity":"bf0ce51f-5328-4398-bfe2-d5170ab3bcb7","order_by":1,"name":"Juan Tamayo","email":"","orcid":"","institution":"University of California, Davis","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Tamayo","suffix":""},{"id":597242826,"identity":"7809abee-24ea-4785-89bf-18c491a55b73","order_by":2,"name":"Kent Pinkerton","email":"","orcid":"","institution":"University of California, Davis","correspondingAuthor":false,"prefix":"","firstName":"Kent","middleName":"","lastName":"Pinkerton","suffix":""},{"id":597242840,"identity":"abfc3c59-170a-45f8-82fc-1b422dfc380e","order_by":3,"name":"Paul Ashwood","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFklEQVRIiWNgGAWjYHACNhDB2C8BJB8wHABxDMCIgSEBv5aZM0BqEkjRsuEGihYG3FrM2Y8/e8xTYSO7+XbzwQ+JP+4wGBw/vPEzT0EdAz97jgE2LZY9OebGPGfSjLfdOZYskZDwjMHgTFqxNI/BYQbJnjdYtRgcyGGTzm07nLjtRo4BUMthBoMbPAaSMwwOABnYbTE4//wZWMvmGfmff0C1GP+cYVDHYI9Ly40EM7CWDRI5bDBbzCQ+GDAzGEjg8MuMN2bSf4B+mXEjzcwiIe0wj+SZtDKLDwaHeSTOPCvAGmL86c8kZwBDrH9G8uMbH2wOy/EdP7z5RsKfOjn+9uQNWB2GLsCDwSCoZRSMglEwCkYBBgAA6CVoYzfflkcAAAAASUVORK5CYII=","orcid":"","institution":"University of California, Davis","correspondingAuthor":true,"prefix":"","firstName":"Paul","middleName":"","lastName":"Ashwood","suffix":""}],"badges":[],"createdAt":"2026-01-30 23:23:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8745297/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8745297/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103621001,"identity":"d606b3e6-86b9-4860-9943-a401516326cf","added_by":"auto","created_at":"2026-02-27 18:13:39","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":356027,"visible":true,"origin":"","legend":"\u003cp\u003eMaternal serum was collected on GD15.5 and run on luminex to analyze cytokine profiles. \u003cem\u003eA. \u003c/em\u003eIn HDM dams, IL-4, IL-5, IL-6, IL-13, G-CSF, IP-10, KC, MIP-1b, TNFa, and RANTES were significantly increased when compared with controls. In HDM+PM dams, IL-5, IL-9, and IP-10 were significantly increased compared with controls. IL-5 was exacerbated in the HDM+PM group compared to HDM alone.\u003cem\u003e B and C. \u003c/em\u003eCytokine levels in maternal serum at GD15.5. \u003cem\u003eD. \u003c/em\u003ePCA and cytokine loadings of maternal serum cytokine data. \u003cem\u003eD. \u003c/em\u003eCorrelation matrices of maternal serum cytokines. \u003cem\u003eE. \u003c/em\u003eTissue inflammatory scoring in maternal lungs showed increased histopathology scoring in HDM and HDM+PM in the alveolar, pleural, perivascular, bronchiolar, and overall extent. The alveolar region showed exacerbated scoring in the HDM+PM compared to HDM alone. P-values are denoted as described in prism: *0.05, **0.001, ***0.0001 (N=10-14 per group).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8745297/v1/9e3faac23ccbc68d695f2140.jpeg"},{"id":103621005,"identity":"a66fd9bf-3310-4010-a36c-820d5680a02b","added_by":"auto","created_at":"2026-02-27 18:13:39","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":428776,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eA. \u003c/em\u003eIn the placenta of HDM+PM offspring, IL-4, IL-5, and KC were significantly increased. IL-5 and IP-10 were increased in the HDM alone group.\u003cem\u003e B.\u003c/em\u003e Heat map p values of all cytokines within range in PBS vs HDM, PBS vs HDM+PM, and HDM vs HDM+PM. \u003cem\u003eC\u003c/em\u003e. PCA analysis and cytokine loadings of placenta cytokine data. \u003cem\u003eD.\u003c/em\u003eCorrelation plots showing pearson coefficients between all in-range cytokines. P-values are denoted as described in prism: *0.05, **0.001, ***0.0001 ****\u0026lt;0.0001 (n=10 per group).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8745297/v1/6b8477a567153fc4db94b4ee.jpeg"},{"id":104399627,"identity":"7b6c9d60-e414-4900-98a8-c439337ae1b1","added_by":"auto","created_at":"2026-03-11 12:06:59","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":368406,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eA. \u003c/em\u003eIn the placenta of HDM+PM offspring, IL-4, IL-5, and KC were significantly increased. IL-5 and IP-10 were increased in the HDM alone group.\u003cem\u003e B.\u003c/em\u003e Heat map p values of all cytokines within range in PBS vs HDM, PBS vs HDM+PM, and HDM vs HDM+PM. \u003cem\u003eC\u003c/em\u003e. PCA analysis and cytokine loadings of placenta cytokine data. \u003cem\u003eD.\u003c/em\u003eCorrelation plots showing pearson coefficients between all in-range cytokines. P-values are denoted as described in prism: *0.05, **0.001, ***0.0001 ****\u0026lt;0.0001 (n=10 per group).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8745297/v1/de50e6e777ef08f2dd1e8d7f.jpeg"},{"id":104399149,"identity":"2321113a-a523-4a3e-9c28-f55acf3eb72e","added_by":"auto","created_at":"2026-03-11 12:04:50","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":394978,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eA and B. \u003c/em\u003eIn the cortex of p15 HDM+PM and PM offspring, IFNg was significantly decreased. MIP-2 was increased in HDM+PM when compared to controls, HDM alone, and PM alone. \u003cem\u003eC\u003c/em\u003e. PCA and cytokine loadings of p15 cortical cytokine data. \u003cem\u003eD.\u003c/em\u003eCorrelation plots showing pearson coefficients between all in-range cytokines. P-values are denoted as described in prism: *0.05, **0.001, ***0.0001 ****\u0026lt;0.0001 (n=10-14 per group).\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8745297/v1/9a7d535122d0d6a15d340d08.jpeg"},{"id":104399279,"identity":"dc0275c3-33a6-4249-bf97-ff779dba8027","added_by":"auto","created_at":"2026-03-11 12:05:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":25269,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eA and B. \u003c/em\u003eIL-4 and G-CSF were significantly decreased in the cerebellum of HDM+PM offspring. \u003cem\u003eC\u003c/em\u003e. PCA and cytokine loadings of p15 cerebellar cytokine data. \u003cem\u003eD.\u003c/em\u003eCorrelation plots showing pearson coefficients between all in-range cytokines. P-values are denoted as described in prism: *0.05, **0.001, ***0.0001 ****\u0026lt;0.0001 (n=10-14 per group).\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8745297/v1/5b68c50a3df0f2ea348064eb.png"},{"id":104399202,"identity":"d67e2b26-1fd6-4c1b-bf16-ec1cc8c0541a","added_by":"auto","created_at":"2026-03-11 12:05:04","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":415592,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eA and B. \u003c/em\u003eIn the hippocampus of both HDM and HDM+PM offspring, IL-6, IL-9, and TNFa were significantly elevated compared with controls and IP-10, IL-10, IL-1b, and IL-1a were significantly decreased. KC was decreased only in the HDM+PM group compared to controls. There were no significant changes in the PM offspring. \u003cem\u003eC\u003c/em\u003e. PCA and cytokine loadings of p15 hippocampal cytokine data. \u003cem\u003eD.\u003c/em\u003e Correlation plots showing pearson coefficients between all in-range cytokines. \u003cem\u003eE\u003c/em\u003e. Microglial density was significantly higher in the HDM+PM group compared to controls in the p15 hippocampus. P-values are denoted as described in prism: *0.05, **0.001, ***0.0001 ****\u0026lt;0.0001 (n=10-14 per group for Luminex and 5 per group for microglial density).\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8745297/v1/39f9a145ced967f1b70c7867.jpeg"},{"id":104834954,"identity":"9fc069de-501f-45a6-8edb-85f941cd46cc","added_by":"auto","created_at":"2026-03-17 17:36:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2729428,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8745297/v1/afc3af4d-2627-4ff6-82ec-79227efa4617.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A Novel Model of House Dust Mite-Induced Maternal Allergic Asthma and Neuroinflammation in the Offspring","fulltext":[{"header":"Background","content":"\u003cp\u003eAutism Spectrum Disorder (ASD) is a neurodevelopmental disorder that has increased in prevalence, with 1 in 31 children in the US being diagnosed by the age of 8 (Shaw et al., 2025). While the underlying etiology of ASD is unknown, several genetic and environmental factors have been associated with an increased risk of ASD (Goines \u0026amp; Ashwood, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Sealey et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). During fetal neurodevelopment, maternal inflammation can have lasting impacts on offspring brain function that can lead to neuroinflammation and behavioral alterations. For instance, maternal infections that resulted in hospitalization during early pregnancy were significantly associated with birthing a child later diagnosed with ASD (Atladottir et al., 2010). Animal models have further demonstrated this effect by using viral and bacterial mimics to assess altered neurodevelopment, behavior, and neuroinflammation in the offspring (Choi et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Haddad et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Schwartzer et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Sim\u0026otilde;es et al., 2018). In addition, chronic inflammatory conditions during pregnancy are also linked with an increased risk of ASD (Croen et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMaternal asthma has been identified as the most common maternal immune condition, to increase risk for ASD in population-based studies (Patel et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Croen et al., 2024; Croen et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Asthma is a T-helper type 2 (T\u003csub\u003eH\u003c/sub\u003e2) driven chronic inflammatory condition typically characterized by an increase in IL-4, IL-5, and IL-13, along with immune cell infiltration of the airways (Gans \u0026amp; Gavrilova, 2019). During pregnancy, asthma symptoms can become exacerbated and lead to an increase in hospital visits and hospitalization (Abdullah et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Furthermore, animal models of ovalbumin (OVA) induced maternal allergic asthma (MAA) have shown an increase in marble burying behavior and a decrease in social interactions in offspring of exposed dams (Schwartzer et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Church et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In the same model, offspring showed increased pro-inflammatory cytokines in the fetal whole brain (Tamayo et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Increased neuroinflammation was also seen in postnatal day (p)15 and p35 hippocampus and cortical regions (Tamayo et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile a useful tool for animal models of asthma, OVA is not the most physiologically relevant asthma causing agent in humans. House dust mite (HDM) is a common allergen associated with asthma in humans, and many studies are shifting towards using natural allergens, like HDM, to induce asthma in animal models (Casta\u0026ntilde;eda \u0026amp; Pinkerton, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). However, HDM has not yet been used in mouse models to study MAA and effects on offspring neurodevelopment. Particulate matter (PM) exposure has been shown to exacerbate asthma symptoms caused by HDM (Casta\u0026ntilde;eda \u0026amp; Pinkerton, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In the environment, PM can vary greatly in composition, while exposed individuals can vary in terms of the time of onset and duration of exposure (Casta\u0026ntilde;eda \u0026amp; Pinkerton, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Carosino et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Even if only introduced during the sensitization phase of asthma, PM can exacerbate the hallmarks of asthma such as cellular infiltration, airway inflammation, and oxidative stress (Carosino et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). PM exposure has also been linked to an increased risk of ASD (Talbott et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDue to these associations, this study aimed to investigate whether PM exacerbation of MAA during the sensitization period led to altered neuroinflammatory profiles in the offspring. We used a novel experimental model of HDM-induced MAA and pre-pregnancy exposure to PM collected from the central valley in California to determine the effect of each exposure, separately and in combination compared with PBS controls, on maternal responses during pregnancy and the offspring cytokine profiles in the brain. We hypothesized that exposure to PM during the sensitization phase would exacerbate maternal asthma and lead to increased inflammation in offspring brains in HDM\u0026thinsp;+\u0026thinsp;PM groups.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eBALB/c mice from Envigo Laboratories (Livermore, CA) were housed with same sex littermates at the Center for Health and the Environment at University of California, Davis in Davis, CA. Mice were kept in a 23\u003csup\u003eo\u003c/sup\u003eC room with a 12-hour light/dark cycle and food and water provided as needed. Procedures and protocols were performed with approval by the University of California, Davis Institutional Animal Care and Use Committee and following the National Institute of Health Guide for the Care and Use of Laboratory Animals.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMaternal allergic asthma and particulate matter exposure paradigm\u003c/h3\u003e\n\u003cp\u003eAll mice were purchased at 7\u0026ndash;8 weeks of age. After allowing mice to adjust to their environment for one week after purchasing, female mice were anesthetized with isoflurane and sensitized intranasally with 25 \u0026micro;l of 1 mg/mL HDM resuspended in phosphate buffered saline (PBS) three times in one week, every other day. Mice in the PM and HDM\u0026thinsp;+\u0026thinsp;PM groups were also exposed intranasally to 33.3 \u0026micro;l of 1 mg/mL PM. Mice were then paired for mating with two females per one male and checked daily for the presence of a seminal plug. Presence of a seminal plug was noted as gestational day (GD) 0.5. On gestational days 12.5 and 15.5, asthma was induced in pregnant females by intranasally exposing dams to 25 \u0026micro;g of HDM, in the HDM and HDM\u0026thinsp;+\u0026thinsp;PM groups. The PBS control group or PM alone group were given intranasal PBS during the asthma induction phase.\u003c/p\u003e\n\u003ch3\u003eTissue collection and lysis\u003c/h3\u003e\n\u003cp\u003eFour hours following the final exposure on GD 15.5, a subset of dams from each group were euthanized using CO\u003csub\u003e2\u003c/sub\u003e for collection of maternal serum and lung tissue. The remaining dams were left undisturbed to give birth naturally. On postnatal (p) day 15, offspring were anesthetized with isoflurane and underwent a terminal cardiac perfusion with ice cold PBS. Following perfusion, brains were collected and micro dissected to separate the cortex, cerebellum and hippocampus. Brain tissue was flash frozen and stored at -80\u003csup\u003eo\u003c/sup\u003eC for future use. Tissue was lysed using cell lysis buffer (Cell Signaling, Danvers MA) and protein from the tissue lysate was quantified using a Bradford assay.\u003c/p\u003e\n\u003ch3\u003eLung collection and analysis\u003c/h3\u003e\n\u003cp\u003eAt necropsy, the trachea of the maternal mice was cannulated, the right mainstem bronchus ligated, and right lobes removed, while the left lung was inflation-fixed with 4% paraformaldehyde at a hydrostatic pressure of 30 cm for one hour. The left lung was placed for 24 hours in fixative prior to transfer into 70% ethanol for storage. The fixed left lung was subsequently cut into four transverse sections, dehydrated in a graded series of ethanol and xylene, and embedded in paraffin. A rotary microtome was used to prepare 5 \u0026micro;m thick tissue sections stained with hematoxylin and eosin for viewing. Lung tissue sections were evaluated using a semi-quantitative grading system for inflammation of the airways, blood vessels, alveoli, and pleura. This scoring system evaluated the extent (0 to 3) and severity (0 to 3) of inflammation for each region of the lungs.\u003c/p\u003e\n\u003ch3\u003eCytokine and Chemokine analysis\u003c/h3\u003e\n\u003cp\u003eCytokines and chemokines in maternal serum and offspring tissue lysates were measured using a magnetic bead 25-plex immunoassay (Milliplex Mouse Cytokine/Chemokine Magnetic Bead Panel #MCYTMAG70PMX25BK). The following cytokines, chemokines, and growth factors were analyzed: granulocyte colony stimulating factor (G-CSF), granulocyte colony stimulating factor (GM-CSF), IFN-γ, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17, interferon gamma-induced protein 10 (IP-10), keratinocyte chemoattractant (KC), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1 alpha (MIP-1α), MIP-1β, MIP-2, chemokine ligand 5 (CCL5/RANTES), and tumor necrosis factor alpha (TNF-α). The plate was washed prior to adding standards and samples following the manufacturer\u0026rsquo;s protocol. For maternal serum, 25 \u0026micro;l of serum was loaded onto the plate. For brain tissue lysates, protein concentration was normalized so that 100 \u0026micro;g of protein was added to each well in duplicate, along with appropriate standards and quality controls. Antibody-coupled magnetic beads were added to each well and incubated overnight. After incubation, the plate was washed following the manufacturer\u0026rsquo;s protocol and then incubated with biotinylated detection antibody, and then with streptavidin-phycoerythrin. The plates were read on a Bio-Rad Bio-plex 200 plate reader (Bio-Rad Laboratories, Hercules, CA). The following are the minimum levels of detection for each analyte: G-CSF: 1.7 pg/mL; GM-CSF: 10.9 pg/mL; IFNγ: 1.1 pg/mL; IL-1α: 10.3 pg/mL; IL-1β: 5.4 pg/mL; IL-2: 1.0 pg/mL; IL-4: 0.4 pg/mL; IL-5: 1.0 pg/mL; IL-6: 1.1 pg/mL; IL-7: 1.4 pg/mL; IL-9: 17.3 pg/mL; IL-10: 2.0 pg/mL; IL-12 (p40): 3.9 pg/mL; IL-12 (p70): 4.8 pg/mL; IL-13: 7.8 pg/mL; IL-15: 7.4 pg/mL; IL-17: 0.5 pg/mL; IP-10: 0.8 pg/mL; KC: 2.3 pg/mL; MCP-1: 6.7 pg/mL; MIP-1α: 7.7 pg/mL; MIP-1β: 11.9 pg/mL; MIP-2: 30.6 pg/mL; RANTES: 2.7 pg/mL; TNF-α: 2.3 pg/mL. If more than 60% of samples fell out of range they were excluded from analysis. For cytokines with only a few samples out of range, the sample was given a proxy value of one half the minimum level of detection for that analyte.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMicroglia density analysis\u003c/h2\u003e \u003cp\u003eFor analysis of microglial density, in the hippocampus corresponding to region 72 in the Allen Brain Atlas, we closely followed a protocol used previously in our studies (Tamayo et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Briefly, after perfusion, right hemispheres were fixed in PFA, then transferred to a sucrose gradient. They were sliced using a cryostat into 20um sections. The 6th well of sections was used for IBA1 staining using DAB substrate. The sections were imaged at 20x on a brightfield microscope (Nikon Eclipse Ci, Nikon, Tokyo) and merged using imageJ. Merged images were then analyzed using an imageJ script outlined in Marques et al., 2023. Microglia were also manually scored by an unbiased and blinded laboratory member to confirm accuracy of our semi-automated scoring.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analysis was performed using GraphPad Prism v10.2.0 (GraphPad Software, Boston, MA). Outliers Identity was performed via ROUT and testing was performed on data excluding any outliers. One-way ANOVA with multiple comparisons and Sidak adjustment for multiple comparisons was run to determine statistical significance with \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05 determining significance. Descriptive statistics including mean, median, and interquartile range were also obtained. Heat maps were made using the p-values obtained from the ANOVA. We also ran principal component analysis (PCA), selecting principal components based on parallel analysis with a percentile level of 95%. Symbol fill color was determined by the categorical variable, which was treatment group. Cytokines were used as continuous variables. For correlation analyses, data was separated by treatment group and r was computed for every pair of Y data sets, creating a correlation matrix. This data shows how patterns of cytokines vary in each sample in relation to other cytokines, and reveals valuable information that may not show up on statistical analyses of cytokine levels alone. Microglia counts were analyzed using one-way ANOVA with adjustment for multiple comparisons.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMaternal lung inflammation\u003c/h2\u003e \u003cp\u003eSemi-quantitative analysis of the maternal lungs showed no significant differences between the PBS control mice or the PM-sensitized mice in the airways, blood vessels, alveoli, or pleura. In contrast, significant differences were found in the degree of inflammation for all four regions in the HDM and HDM\u0026thinsp;+\u0026thinsp;PM exposure groups. One notable finding was a statistically significant increase in the degree of alveolar inflammation in the HDM\u0026thinsp;+\u0026thinsp;PM group in comparison to the HDM-only group (\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0335).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMaternal Serum Cytokine Analysis\u003c/h2\u003e \u003cp\u003eSerum cytokines were measured at GD15.5 in dams to determine systemic inflammatory responses in each treatment group. A significant increase in T\u003csub\u003eH\u003c/sub\u003e2 cytokines was seen in the maternal serum in HDM and HDM\u0026thinsp;+\u0026thinsp;PM groups compared to PBS controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). In HDM and HDM\u0026thinsp;+\u0026thinsp;PM dams, IL-5 (HDM \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0345; HDM\u0026thinsp;+\u0026thinsp;PM \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and IP-10 (HDM \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; HDM\u0026thinsp;+\u0026thinsp;PM \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0002) were increased, with IL-5 more significantly increased in the combined exposure group than HDM alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). IL-4 was increased in the HDM dams (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0049) and trended towards increase in the HDM\u0026thinsp;+\u0026thinsp;PM dams (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0550) compared to PBS alone but did not reach significance after corrections for multiple comparisons. The PM alone group did not have an inflammatory effect on maternal cytokine responses when given antenatally in this model. Overall, combined with data from lung tissue, this shows that our model of HDM allergic asthma induced a T\u003csub\u003eH\u003c/sub\u003e2 skewed, inflammation, and asthma-like condition in the dams.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOn PC analysis, several data points from the HDM\u0026thinsp;+\u0026thinsp;PM group clustered away from the rest of the data, but no other apparent separations were seen. Correlation analysis shows a shift towards strong negative correlations in the PM group. There was increased strength of positive correlations between cytokines like IL-13, IL-15, and IL-17 in the HDM\u0026thinsp;+\u0026thinsp;PM group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Overall, correlation analysis shows us several patterns between cytokines that were otherwise not significant, especially the changes to the PM group, as none were seen in mean differences in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eGD15.5 placental cytokine profiles\u003c/h2\u003e \u003cp\u003eIn the placenta, an overall increased T\u003csub\u003eH\u003c/sub\u003e2 cytokine profile was seen, with IL-5 increased in both HDM (\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0326) and HDM\u0026thinsp;+\u0026thinsp;PM (\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0053) offspring and IL-4 increased in only the HDM\u0026thinsp;+\u0026thinsp;PM offspring (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Increased KC in the HDM\u0026thinsp;+\u0026thinsp;PM offspring compared to the PBS controls was also observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter performing PC analysis, HDM\u0026thinsp;+\u0026thinsp;PM data separated from PBS and HDM along PC2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). The PC1 accounted for 40.1% of variance between data and PC2 accounted for 19.92% of variance between data. Furthermore, comparing cytokines using a correlation matrix, several important differences were noted between the treatment groups. PBS placentas were on the whole positively correlated, with no strong correlation between T\u003csub\u003eH\u003c/sub\u003e2 cytokines. The strongest correlation in the PBS group was seen between IL-1α and TNFα. In the HDM and HDM\u0026thinsp;+\u0026thinsp;PM groups, positive correlations were seen between the T\u003csub\u003eH\u003c/sub\u003e2 cytokines IL-4 and IL-5, which were both significantly elevated in the placenta (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). In HDM\u0026thinsp;+\u0026thinsp;PM, a strong correlation between these T\u003csub\u003eH\u003c/sub\u003e2 cytokines and IL-6 was also observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). When analyzed separately, male and female offspring did not have significant differences (data not shown).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eGD15.5 fetal brain cytokine profiles\u003c/h2\u003e \u003cp\u003eIn the fetal brain, decreased G-CSF and MCP-1 was observed in the HDM (G-CSF \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001; MCP-1 \u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0472) and HDM\u0026thinsp;+\u0026thinsp;PM offspring (G-CSF \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.0001; MCP-1 \u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0023). In addition, HDM\u0026thinsp;+\u0026thinsp;PM offspring had decreased MIP-2 (\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0119) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePCA showed a clear separation of PM from the other treatment groups along PC2 with PC1 accounted for 42.21% and PC2 accounts for 17.77% of the variance of the data (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Correlation matrices showed several differences between treatment groups. In the PBS offspring, IL-1α was positively correlated with MCP-1, IL-9, and IP-10; and IL-10 was positively correlated with MIP-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). In contrast, PM offspring showed a negative correlation between IL-1β and MIP-1α. In HDM, IL-1α was correlated with IL-1β, IL-2, IL-10, and MIP-1α. The HDM\u0026thinsp;+\u0026thinsp;PM offspring had strong positive correlations between G-CSF and MCP-1, IL-2, IL-1α, and IL-9; in addition, IL-9 correlated with IL-10 and IL-1β; and, the chemokine MCP-1 correlated with IP-10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Taken together these correlations showed a distinct pattern of cytokines in the fetal brain depending on the mother\u0026rsquo;s treatment group. There were no significant differences between male and female fetal brain cytokines (data not shown).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ep15 Cortex cytokine profiles\u003c/h2\u003e \u003cp\u003eIn the cortex, PM alone showed a significant decrease in IFNg (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0120) compared to controls. In HDM alone, there were no significant changes when compared to controls. In the combined HDM\u0026thinsp;+\u0026thinsp;PM exposure group, significantly decreased IFNg was found, and there was also increased MIP-2 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0077), compared to both PBS controls and the HDM alone group. IL-17 trended towards increased levels in HDM\u0026thinsp;+\u0026thinsp;PM (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0501).\u003c/p\u003e \u003cp\u003eThe cortical cytokine data suggested a combined effect of HDM and PM on the offspring brain. There was no obvious separation between groups upon PC analysis of PC1 and PC2. PC1 accounted for 30.93% of variance in data and PC2 accounted for 19.52% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). When investigating correlations between significant cytokines, no strong correlations were observed with IFNg in any treatment groups. MIP-2 was correlated with GM-CSF in PBS, but was not strongly correlated with any cytokines in the other treatment groups. IL-17 was positively correlated with IL-13 in PBS controls, and with IL-6 in PM, and with IP-10 in HDM\u0026thinsp;+\u0026thinsp;PM. Interestingly, IL-6 and KC were positively correlated in both PBS and HDM\u0026thinsp;+\u0026thinsp;PM offspring (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ep15 Cerebellum cytokine profiles\u003c/h2\u003e \u003cp\u003eIn the cerebellum there were no significant changes seen in cytokine levels in HDM alone or PM alone groups compared to PBS controls in the p15 offspring. However, significantly decreased levels of IL-4 (p\u0026thinsp;=\u0026thinsp;0.0424) and G-CSF (p\u0026thinsp;=\u0026thinsp;0.0048) were observed in the HDM\u0026thinsp;+\u0026thinsp;PM group when compared to PBS controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThere was no separation between groups along the PC2 axis, with only a slight skewing of the HDM\u0026thinsp;+\u0026thinsp;PM data points along the PC1 axis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). IL-4, which was significantly decreased in the cerebellum of p15 offspring, was positively correlated with IL-2 and IL-1α. IL-4 was also correlated with IL-2 in both PM and HDM\u0026thinsp;+\u0026thinsp;PM group, but was not significantly associated in HDM. In the HDM\u0026thinsp;+\u0026thinsp;PM offspring, IL-4 was positively correlated with IL-6 and IL-15 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). The data showed an overall decrease in regulation and growth factor production in the cerebellum in the dual exposure group, but with no major impact of either of the single exposure groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ep15 Hippocampus cytokine profiles\u003c/h2\u003e \u003cp\u003eIn the hippocampus of both HDM and HDM\u0026thinsp;+\u0026thinsp;PM offspring, significantly increased IL-6 (HDM \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0004; HDM\u0026thinsp;+\u0026thinsp;PM \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001), IL-9 (HDM \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0005; HDM\u0026thinsp;+\u0026thinsp;PM \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0206), and TNFα (HDM \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001; HDM\u0026thinsp;+\u0026thinsp;PM \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001) was observed (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). In contrast, IL-10 (HDM \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001; HDM\u0026thinsp;+\u0026thinsp;PM \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001), IP-10 (HDM p\u0026thinsp;=\u0026thinsp;0.0187; HDM\u0026thinsp;+\u0026thinsp;PM p\u0026thinsp;=\u0026thinsp;0.0079), IL-1α (HDM \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0053; HDM\u0026thinsp;+\u0026thinsp;PM \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0027), and IL-1β (HDM \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0027; HDM\u0026thinsp;+\u0026thinsp;PM \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0006) were significantly decreased in both HDM and HDM\u0026thinsp;+\u0026thinsp;PM groups compared to the PBS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). KC levels were decreased only in HDM\u0026thinsp;+\u0026thinsp;PM (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0164) compared to PBS controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). These data show significantly altered inflammatory and anti-inflammatory cytokine profiles in both HDM and HDM\u0026thinsp;+\u0026thinsp;PM offspring, with a larger effect seen in the combined exposure group for some cytokines.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePC analysis shows clear separation of HDM and HDM\u0026thinsp;+\u0026thinsp;PM groups from PBS and PM alone groups along the PC1 axis. Some separation between HDM and HDM\u0026thinsp;+\u0026thinsp;PM also occurred along the PC2 axis. PC1 in the hippocampus was responsible for 31.26% of variance in the data and PC2 was responsible for 29.8% (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). In the PBS group, cytokines seen as significant in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb were positively correlated with each other. However, when examining the PM and HDM data, these positive correlations were almost completely absent. In the HDM\u0026thinsp;+\u0026thinsp;PM group, no cytokines were significantly correlated with each other, positively or negatively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). In addition, no sex differences in cytokines were observed after adjustments for multiple comparisons.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003ep15 Hippocampal microglia density\u003c/h2\u003e \u003cp\u003eAs we saw the most alterations in the hippocampal cytokines, we chose to do additional analysis on the microglial density. Moreover, prior work in our OVA models showed changes in microglia only in the hippocampus at p15. Allen brain atlas section 72 was used as reference to choose sections for analysis. After using a semi- automatic method for cell counting and manual counting by an unbiased and blinded laboratory member, the density of microglia was determined. In this region of the hippocampus significantly decreased microglia density was observed in the HDM\u0026thinsp;+\u0026thinsp;PM group when compared to the PBS group (\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0420) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). PM and HDM alone did not yield any significant differences when compared to PBS controls.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eMaternal asthma and exposure to PM exposure have both been linked with an increased risk of neuroinflammation in the offspring (Croen et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Tamayo et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Talbott et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Previous studies have used an OVA-based regimen to model MAA, while in the current study the more physiologically relevant allergen HDM was used to induce an asthma response in the dams. Given that PM can aggravate asthma during sensitization, we hypothesized that the addition of PM would exacerbate maternal systemic inflammation in the dams and increase neuroinflammatory responses in the offspring brain. We saw an additive effect of PM on maternal asthma induction with elevated T\u003csub\u003eH\u003c/sub\u003e2 cytokine in the maternal serum and in the alveolar region of the lungs. In addition, when analyzing brain samples taken from p15 offspring there was an increased additive effect on cytokine levels when dams were exposed to both PM and HDM.\u003c/p\u003e \u003cp\u003eAllergic asthma is associated with a T\u003csub\u003eH\u003c/sub\u003e2 immune response. Asthma is a highly heterogeneous disease, with subtypes of eosinophilic (T\u003csub\u003eH\u003c/sub\u003e2 driven), neutrophilic (T\u003csub\u003eH\u003c/sub\u003e17 driven), or combined (eosinophilic\u0026thinsp;+\u0026thinsp;neutrophilic) (Gans \u0026amp; Gavrilova, 2019). Maternal serum data confirmed a systemic maternal inflammatory response in the HDM and HDM\u0026thinsp;+\u0026thinsp;PM groups. The cytokines seen in maternal serum suggested a T\u003csub\u003eH\u003c/sub\u003e2 response in HDM exposure groups due to increased IL-4, IL-5, and IL-6. The cytokines IL-4, IL-5, and IL-13 are commonly elevated in asthma patients (Lambrecht et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) with IL-6 also seen in T\u003csub\u003eH\u003c/sub\u003e2 immune reactions (Wong et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Furthermore, we saw increased lung inflammation in both HDM and HDM\u0026thinsp;+\u0026thinsp;PM groups, consistent with allergic response. Interestingly, both maternal sera and lung data showed an additive increased inflammatory response in the HDM\u0026thinsp;+\u0026thinsp;PM group over the HDM alone, suggesting PM primed a more exaggerated maternal response.\u003c/p\u003e \u003cp\u003eThe placenta is the primary source of communication between the mother and developing fetus. Cytokines can be produced by placental cells, especially placental macrophages, and can alter the placental microenvironment, potentially leading to changes in fetal development (Ben-Rafael \u0026amp; Orvieto et al., 1992). During the entire pregnancy, but increasingly so in the third trimester in humans, cytokine profiles in the placenta skew towards T\u003csub\u003eH\u003c/sub\u003e2, which helps prevent rejection of the fetus (Dealtry et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Doria et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). However, pregnancy complications have also been noted to occur with dominant T\u003csub\u003eH\u003c/sub\u003e2 responses. In this model, we saw a significant increase in the T\u003csub\u003eH\u003c/sub\u003e2 cytokines such as IL-4 and IL-5 in the placenta of HDM and HDM\u0026thinsp;+\u0026thinsp;PM offspring. This mimics the pattern seen in maternal serum. Further research on isolated placental cells including macrophages which are major producers of cytokines in the placenta and participate in not only immunity but regulation of other cells in the placenta and placental morphology and formation (Pavlov et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) is warranted. Identifying signaling pathways associated with PM and HDM exposures may also reveal how changes in cytokine profiles can impact the fetal microenvironment and help connect the maternal, placental and fetal brain data.\u003c/p\u003e \u003cp\u003eCytokines are an important part of normal brain development during gestation and post-natally. Cytokines are crucial to neurodevelopment at homeostasis, and deviations from these levels, especially during key neurodevelopmental windows, have been associated with disrupted neurodevelopment (Ratnayake et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Dziegielewska et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). In the fetal brain data decreases in 3 cytokines/chemokines were observed in the dual exposure group: MIP-2, MCP-1, and G-CSF. The chemokine MIP-2, is a neutrophil chemoattractant (Driscoll, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) and has been associated with traumatic brain injury or hypoxic conditions. However, little research has been done on whether MIP-2 can play a role in development during early neurodevelopmental windows. Altered MCP-1 levels have been noted in several human and rodent studies linked with ASD or schizophrenia, with increased levels observed in ASD individuals (Maldonado-Ruiz et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zerbo et al., 2014; Frydecka et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This is in contrast to the fetal brain data form this model and may reflect dynamic changes over time and development (Robertson et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). The innate cytokine, G-CSF, been seen in other animal models with relevance to ASD and higher levels improved symptoms of ASD in rodent studies (Durankus et al., 2022). Mechanistically, G-CSF can interact with NMDA receptors to potentially ameliorate ASD associated behaviors (Mishra et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In the MAA model, a decrease in neuroprotective G-CSF may mean a loss in NMDA receptor modulation critical for typical development.\u003c/p\u003e \u003cp\u003eThe cortex consists of gray matter that exhibits altered function and connectivity in ASD patients (Jawabri \u0026amp; Sharma, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Gandal et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Kana et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Tamayo et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In the cortex of HDM\u0026thinsp;+\u0026thinsp;PM mice, two cytokines were significantly altered compared to the controls. One of the cytokines, IFNγ, was decreased in the HDM\u0026thinsp;+\u0026thinsp;PM group. and is involved in regulating social behavior. In a mouse model using an IFNγ knockout, IFNγ\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice showed social deficits and altered neural circuits (Filiano et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The second differentially produced cytokine/chemokine, MIP-2, was significantly increased in the cortex of the HDM\u0026thinsp;+\u0026thinsp;PM group. MIP-2, together with KC, can induce astrocytes to increase expression of chemokines like RANTES, IP-10, and the cytokine IL-6 (Luo et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). In the cerebellum, IL-4, was significantly decreased in offspring of the HDM\u0026thinsp;+\u0026thinsp;PM dams. In the brain, IL-4 is typically seen as regulatory and can rescue social behaviors (Derecki et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). IL-4 can modulate microglia, skewing them towards an anti-inflammatory state (Zhao et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). During the critical p15 developmental window, when synaptic pruning activity peaks, regulatory cytokines play an important role in modulating microglial activity. A decrease in these regulatory cytokines may contribute to increased neuroinflammation and over-pruning of synapses. The cerebellum is involved in motor function and learning, but also in several emotional and cognitive processes (Schmahmann \u0026amp; Caplan, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). In both the cortex and cerebellum, cytokines are only significant in the HDM\u0026thinsp;+\u0026thinsp;PM group, with several effects exacerbated compared to both PBS and HDM groups. While many changes with just HDM exposure alone are seen, differing patterns and exacerbations of allergic asthma effects by PM are evident on the offspring brain at p15.\u003c/p\u003e \u003cp\u003eIncreased IL-6 in the brain was associated with increased excitatory synapses in the hippocampus and somatosensory cortex of mice, as well as ASD-like behaviors (Wei et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In this model, IL-6 was increased in maternal serum and the p15 hippocampus in offspring after HDM or HDM\u0026thinsp;+\u0026thinsp;PM exposures. The hippocampus is involved in social behavior, learning, memory, and spatial reasoning in the brain (Immordino-Yang \u0026amp; Singh, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Rubin et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Banker et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Altered shape, size, and function of the hippocampus has been seen in ASD individuals (Barnea-Goraly et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Dager et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Solomon et al., 2015). Furthermore, several animal models show neuroinflammation in the hippocampus that was associated with alterations in behaviors, and altered microglia density (Schwartzer et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Tamayo et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The hippocampus is a site of adult neurogenesis, and altered inflammatory profiles may severely impact this throughout development. In humans, IL-6 was increased levels in postmortem cerebellum specimens of ASD patients (Wei et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). KC was the only cytokine in the hippocampus to be increased only in the HDM\u0026thinsp;+\u0026thinsp;PM group compared to HDM alone, or PM alone, or PBS controls. KC is a mouse homolog of the human protein growth-regulated oncogene, and has pro-inflammatory effects (Son et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Levels of hippocampus IL-9, IL-10, and IP-10 were both significantly altered in the HDM and HDM\u0026thinsp;+\u0026thinsp;PM groups. IL-9, which was increased in HDM and HDM\u0026thinsp;+\u0026thinsp;PM groups, has been shown to be increased in PBMC from children with ASD (Ahmad et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and is important in recruitment of T\u003csub\u003eH\u003c/sub\u003e17 cells to the CNS (Zhou et al., 2011). IL-9 can also play a role in neurodevelopment by inhibiting programmed cell death of cortical neurons, a homeostatic process during development (Fontaine et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). IL-10, decreased in HDM and HDM\u0026thinsp;+\u0026thinsp;PM treatment groups, has important immunoregulatory functions that reduce inflammatory responses and regulate function of microglia and astrocytes in the brain. In the absence of IL-10, microglia and astrocytes are known to produce more pro-inflammatory cytokines and reactive oxygen/nitrogen species (Laffer et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhang et al., 2020). Thus, low IL-10 in the HDM\u0026thinsp;+\u0026thinsp;PM exposed group would switch the profile of cytokine messages towards a more inflammatory profile.\u003c/p\u003e \u003cp\u003eIn the HDM\u0026thinsp;+\u0026thinsp;PM group decreased microglial density was observed in the hippocampus at p15, whereas there was no significant effect with HDM alone or PM alone. Fewer microglia in the hippocampus at p15, the peak of synaptic pruning in mice, could suggest this process is altered due to cytokine changes in the region. This can direct future studies, in which we can collect tissue for dendritic spine analysis, pre and post synaptic vesicle density, and interactions between the microglia and synaptic sites. In models of altered neurodevelopment, altered synaptic pruning is seen and associated with worsened behaviors (Kim et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In postmortem ASD brains, increased dendritic spine density is seen, suggesting aberrant pruning (Hutsler \u0026amp; Zhang, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn order to investigate the complex interactions of cytokines PCA and correlative analysis were performed. Along with the impact of significantly increased or decreased cytokines, altered patterns of cytokines may be revealed that were not clear when comparing mean cytokine levels. Altered cytokine ratios may play a significant role in cell function and inflammation. Future studies can then focus not only on the impact of single cytokines on neural cells, but how combinations of cytokines work together. The hippocampus was also the only region that showed clear separation of groups upon principal component analysis. Correlations of cytokines between groups showed a clear positive correlation between cytokines such as IL-9, IL-1α, IL-13, and IL-17. These correlations significantly diminished in both the PM and HDM\u0026thinsp;+\u0026thinsp;PM groups. In regions where few cytokines were seen as significantly changed, we were able to see striking differences in the HDM\u0026thinsp;+\u0026thinsp;PM correlations compared with controls. Changes between the HDM and HDM\u0026thinsp;+\u0026thinsp;PM correlations, in all regions and timepoints, reveal more differences between the groups than seen upon cytokine levels alone.\u003c/p\u003e \u003cp\u003eOverall, the data from this novel model of lung-brain inflammation showed a combined effect of HDM and PM on IL-5 levels in the maternal serum, and in lung inflammation in the alveoli. The maternal exacerbation of asthma then had impacts in the offspring neuroinflammatory environment. In the placenta and fetal brain, an exacerbation of effects in the HDM\u0026thinsp;+\u0026thinsp;PM group over HDM or PM alone was observed. In the hippocampus, altered levels of seven cytokines in both HDM alone and HDM\u0026thinsp;+\u0026thinsp;PM was seen. The hippocampus also showed the most separation of groups in PC analysis, compared with the other regions of the p15 brain. Correlation analysis of all regions of the offspring brain showed changes in cytokine patterns in the HDM\u0026thinsp;+\u0026thinsp;PM group compared to controls, HDM alone, and PM alone.\u003c/p\u003e \u003cp\u003eThe current study several limitations including analysis of a specific PM composition. PM from different geographical regions may vary in the ability to exacerbate asthma and neuroinflammation in the offspring. Only a single time point of serum was taken, and as we do see a combined effect in more cytokines in the offspring brains, it may be plausible that cytokines in the HDM\u0026thinsp;+\u0026thinsp;PM maternal serum had been elevated previously or may increase later in pregnancy. In addition, this study does not include behavioral data. As this is a novel model, behavioral studies should be performed in the future due to MAA being linked to a number of neurodevelopmental disorders. Previously, ASD-like behaviors have been seen in the OVA-induced MAA model (Schwartzer et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis novel model of lung-brain inflammation that was PM presentation of HDM-induced MAA, caused a systemic inflammatory response in the dams which resulted in altered cytokine profiles in placenta, fetal brain, the hippocampus and cortex of p15 offspring. Maternal inflammation followed a T\u003csub\u003eH\u003c/sub\u003e2-like pattern and offspring brains showed altered levels of cytokines that can have profound effects on neurodevelopmental. Several cytokines seen in this study have not been extensively studied in terms of how they may impact microglial function or neurotoxicity. The data showed some evidence for microglia activation in the hippocampus after HDM\u0026thinsp;+\u0026thinsp;PM that warrant further follow up. Future studies should focus on how these changes in cytokines impact neuronal connections and glial cell function.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eASD\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAutism spectrum disorders\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eHDM\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eHouse dust mite\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003ePM\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eParticulate matter\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eMAA\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMaternal allergic asthma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eGD\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGestational day\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003ePBS\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphate buffered saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eP\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePostnatal day\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eIL\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInterleukin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eIFNγ\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInterferon gamma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003ePoly(I\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eC)\u003c/em\u003e:Polyinosinic:polycytidylic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eDAB\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e3,3\u0026rsquo;-Diaminobenzidine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eG-CSF\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGranulocyte colony stimulating factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eGM-CSF\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGranulocyte colony stimulating factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eIP-10\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInterferon gamma-induced protein 10\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eKC\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKeratinocyte chemoattractant\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eMCP-1\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMonocyte chemoattractant protein-1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eMIP-1α\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMacrophage inflammatory protein-1 alpha\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eRANTES\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eChemokine ligand 5\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eTNF-α\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTumor necrosis factor alpha\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eT\u003c/em\u003e\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eT-helper\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eCNS\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCentral nervous system\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eMIA\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMaternal immune activation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003e The studies involved animal models and were reviewed and approved by University of California, Davis Institutional Animal Care and Use Committee (IACUC) and according to guidelines established by National Institute of Health Guide for the Care and Use of Laboratory Animals.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis material is based upon work supported by National Institute of Environmental Health Sciences (R21ES035969) and (R21ES035492), HEDCO foundation, and the Brain Foundation.\u003c/p\u003e \u003cp\u003eAcknowledgements\u003c/p\u003e \u003cp\u003eWe would like to thank Mr. Dale Uyeminami, Dr. Morgan Poindexter, and undergraduate Ryan C. Hart, along with all members of the Ashwood Laboratory and Pinkerton Laboratory for assistance in this project.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eHO. performed experiments, analyzed and interpreted data, and drafted the manuscript. J.T performed experiments. K.P. obtained exposures and implemented exposure panels and lung tissue analysis. P.A. formulated hypothesis, designed the study, edited the manuscript, and provided scientific funding. All authors read and approved of the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdullah K, Zhu J, Gershon A, Dell S, To T. (2020). Effect of asthma exacerbation during pregnancy in women with asthma: a population-based cohort study. 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Granulocyte colony-stimulating factor improved core symptoms of autism spectrum disorder via modulating glutamatergic receptors in the prefrontal cortex and hippocampus of rat brains. ACS Chem Neurosci. 2022;13(20):2942\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatel S, Dale RC, Rose D, Heath B, Nordahl CW, Rogers S, Ashwood P. Maternal immune conditions are increased in males with autism spectrum disorders and are associated with behavioural and emotional but not cognitive co-morbidity. Translational psychiatry. 2020;10(1):286.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePavlov OV, Selutin AV, Pavlova OM, Selkov SA. Two patterns of cytokine production by placental macrophages. Placenta. 2020;91:1\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRatnayake U, Quinn T, Walker DW, Dickinson H. Cytokines and the neurodevelopmental basis of mental illness. Front NeuroSci. 2013;7:180.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRobertson SA, Seamark RF, Guilbert LJ, Wegmann TG. The role of cytokines in gestation. Crit Reviews\u0026trade; Immunol. 1994;14:3\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRubin RD, Watson PD, Duff MC, Cohen NJ. The role of the hippocampus in flexible cognition and social behavior. Front Hum Neurosci. 2014;8:742.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaito S, Nakashima A, Shima T, Ito M. Th1/Th2/Th17 and regulatory T-cell paradigm in pregnancy. Am J Reprod Immunol. 2010;63(6):601\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchmahmann JD, Caplan D. Cognition, emotion and the cerebellum. Brain. 2006;129(2):290\u0026ndash;2.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchwartzer JJ, Careaga M, Onore CE, Rushakoff JA, Berman RF, Ashwood P. 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X.(2020). A1 astrocytes contribute to murine depression-like behavior and cognitive dysfunction, which can be alleviated by IL-10 or fluorocitrate treatment. Journal of neuroinflammation, 17(1), 1\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao X, Wang H, Sun G, Zhang J, Edwards NJ, Aronowski J. Neuronal interleukin-4 as a modulator of microglial pathways and ischemic brain damage. J Neurosci. 2015;35(32):11281\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou, Y., Sonobe, Y., Akahori, T., Jin, S., Kawanokuchi, J., Noda, M., \u0026hellip; Suzumura,A. (2011). IL-9 promotes Th17 cell migration into the central nervous system via CC chemokine ligand-20 produced by astrocytes. The Journal of Immunology, 186(7), 4415\u0026ndash;4421.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-neuroinflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jneu","sideBox":"Learn more about [Journal of Neuroinflammation](http://jneuroinflammation.biomedcentral.com)","snPcode":"12974","submissionUrl":"https://submission.nature.com/new-submission/12974/3","title":"Journal of Neuroinflammation","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Autism, Maternal Immune activation (MIA), Maternal allergic asthma (MAA) Cytokines, regulation, microglia, Lung, Brain","lastPublishedDoi":"10.21203/rs.3.rs-8745297/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8745297/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMaternal immune activation (MIA) through infection, toxic exposure, or chronic inflammatory disease has been linked with an increased risk of neurodevelopmental disorders, in offspring, such as autism spectrum disorder (ASD) or schizophrenia. Previously we have shown that inducing maternal allergic asthma (MAA) with ovalbumin increased pro-inflammatory cytokines during pregnancy and altered behaviors in offspring. Other studies have separately shown that particulate matter (PM) can exacerbate the effects of allergic asthma. In this study, we used house dust mite (HDM) to induce MAA combined with a pre-pregnancy exposure to PM to assess the possible combinatorial effects on maternal inflammation and offspring brain cytokine levels. BALB/c mice were treated preconception with PBS, HDM, PM, or HDM\u0026thinsp;+\u0026thinsp;PM, and subsequently challenged with HDM or PBS on gestational days (GD) 12.5 and 15.5. Maternal serum, placental tissue, and fetal brains were collected on GD15.5. Offspring brains were collected at postnatal day 15 to analyze for differences in cytokine levels across treatments. HDM and HDM\u0026thinsp;+\u0026thinsp;PM treated dams were found to have elevated levels of pro-inflammatory and T-helper (T\u003csub\u003eH\u003c/sub\u003e) type 2 cytokines in the serum and increased inflammation in the lungs, consistent with an allergic response. In the placenta, increased T\u003csub\u003eH\u003c/sub\u003e2 cytokines IL-4 and IL-5 were found in the HDM\u0026thinsp;+\u0026thinsp;PM offspring, consistent with the maternal serum data. Fetal brains had a decrease in MIP-2, MCP-1, and G-CSF in HDM and HDM\u0026thinsp;+\u0026thinsp;PM treatment groups. In the p15 hippocampus, HDM and HDM\u0026thinsp;+\u0026thinsp;PM groups showed increases in several pro-inflammatory cytokines but decreased regulatory cytokine IL-10, representing a skewed neuroinflammatory immune profile. We also saw decreased microglia density in the HDM\u0026thinsp;+\u0026thinsp;PM group in the hippocampus. Altered cytokine profiles were additionally noted in the cortex and cerebellum of p15 offspring. Overall, this data shows an increased pro-inflammatory response and a decreased regulatory response in the brains of offspring in response to maternal immune activation with HDM and PM.\u003c/p\u003e","manuscriptTitle":"A Novel Model of House Dust Mite-Induced Maternal Allergic Asthma and Neuroinflammation in the Offspring","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-27 18:13:34","doi":"10.21203/rs.3.rs-8745297/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-13T05:32:26+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-12T14:50:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-12T05:36:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-04T16:42:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-26T02:34:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"167158517222329752416228795491027753242","date":"2026-02-19T15:30:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"20932149247930825356302525669385682238","date":"2026-02-19T07:36:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"278410780301280969858387280671101048283","date":"2026-02-18T03:08:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"45675679522560927250141472192982811674","date":"2026-02-16T09:28:27+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-16T02:28:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-06T15:34:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-06T11:07:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Neuroinflammation","date":"2026-01-30T23:15:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-neuroinflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jneu","sideBox":"Learn more about [Journal of Neuroinflammation](http://jneuroinflammation.biomedcentral.com)","snPcode":"12974","submissionUrl":"https://submission.nature.com/new-submission/12974/3","title":"Journal of Neuroinflammation","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7d9b6901-4e08-4bff-8d82-70604c724b00","owner":[],"postedDate":"February 27th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-03-13T05:39:19+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-27 18:13:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8745297","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8745297","identity":"rs-8745297","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}