XX sex chromosome complement modulates immune responses to heat-killed Streptococcus pneumoniae immunization in a microbiome-dependent manner

Background Differences in male vs. female immune responses are well-documented and have significant clinical implications. While the immunomodulatory effects of sex hormones are well established, the contributions of sex chromosome complement (XX vs. XY) and gut microbiome diversity on immune sexual dimorphisms have only recently become appreciated. Here we investigate the individual and collaborative influences of sex chromosome complements and gut microbiome bacteria on humoral immune activation. Methods Sham-operated and gonadectomized male and female Four Core Genotype (FCG) mice were immunized with heat-killed Streptococcus pneumoniae (HKSP). Humoral immune responses were assessed, and X-linked immune-related gene expression was evaluated to explain the identified XX-dependent phenotypes. Ex vivo studies investigated the functional role of Kdm6a, an X-linked epigenetic regulatory gene of interest, in mitogenic B cell activation. Additionally, we examined whether gut microbiome communities, or their metabolites, differentially influence immune cell activation in a sex chromosome-dependent manner. Endogenous gut microbiomes were antibiotically depleted and reconstituted with select short-chain fatty acid (SCFA)-producing bacteria prior to HKSP immunization and immune responses assessed. Results XX mice exhibited higher HKSP-specific IgM-secreting B cells and plasma cell frequencies than XY mice, regardless of gonadal sex. Although Kdm6a was identified as an X-linked gene overexpressed in XX B cells, inhibition of its enzymatic activity did not affect mitogen-induced plasma cell differentiation or antibody production in a sex chromosome-dependent manner ex vivo. Enhanced humoral responses in XX vs. XY immunized FCG mice were eliminated after microbiome depletion, indicating that the microbiome contributes to the identified XX-dependent immune enhancement. Reconstituting microbiota-depleted mice with select SCFA-producing bacteria increased humoral responses in XX, but not XY, FCG mice. This XX-dependent enhancement appears to be independent of SCFA production in males, while female XX-dependent responses relied on SCFAs. Conclusions FCG mice have been used to assess the influence of sex hormones and sex chromosome complements on various sexually dimorphic traits. The current study indicates that the gut microbiome impacts humoral responses in an XX-dependent manner, suggesting that the collaborative influence of gut bacteria and other sex-specific factors should be considered when interpreting data aimed at delineating the mechanisms that promote sexual dimorphism.

humoral responses in an XX-dependent manner, suggesting that the collaborative in uence of gut bacteria and other sex-speci c factors should be considered when interpreting data aimed at delineating the mechanisms that promote sexual dimorphism.

Highlights
Humoral immune responses against HKSP immunization are in uenced by the possession of an XX vs. XY sex chromosome complement.
Kdm6a is biallelically expressed in XX B cells and is overexpressed in XX vs. XY-possessing splenocytes and B cells of HKSP-immunized mice.
Inhibition of KDM6a enzymatic activity ex vivo promotes plasma cell differentiation, but not in in a sex chromosome-dependent manner.
XX-dependent immune enhancement is microbiome-dependent, despite minimal differences in microbiome composition or SCFA concentrations being identi ed between XX vs. XY female or XX vs. XY male FCG mice.
Reconstitution of the antibiotic-depleted gut microbiome with select SCFA-producing bacteria enhanced immune responses in XX, but not XY, FCG mice.

Plain English Summary
Male and female immune systems differ in their ability to respond to infectious challenge.While males tend to be more susceptible to infection and produce lower amounts of antibodies in response to vaccination, females are more prone to develop autoimmune and in ammatory diseases.Key contributors to these differences include sex hormones, sex chromosome complement (XX in females vs. XY in males), and distinct gut microbial communities capable of regulating immune activation.While each factor has been studied individually, this research underscores the potential for these factors to collaboratively impact immune activation.Here, possession of an XX vs. XY sex chromosome complement was demonstrated to enhance antibody responses to heat-killed Streptococcus pneumoniae vaccination.While attempting to determine the underlying cause of this immune enhancement, the gut microbiome was identi ed to play a critical role.In the absence of an intact gut microbiome, XX immune activation was reduced to levels similar to those seen in XY sex chromosome complement-possessing mice.Replacement of the depleted gut microbiomes with select bacterial species rescued the XXdependent immune enhancement.Further studies are needed to determine exactly how these microbial communities impact immune activation in a sex chromosome complement-dependent manner.Our ndings highlight the need to consider the collaborative effects of individual sex-speci c factors when attempting to understand immune sex biases, as a better understanding of these interactions will likely pave the way for improving therapeutics and vaccines tailored to both sexes.

Background
Sex differences in immune responses have been well characterized (1).In general, females elicit stronger humoral and cell-mediated responses to infection and respond better to vaccination than males, but in turn are more susceptible to autoimmune and in ammatory disorders (1)(2)(3)(4)(5)(6)(7).Historically, hormonal differences have been described as the predominant determinant contributing to such sexual dimorphisms, as estradiol and progesterone are known to be immunostimulatory in nature and testosterone immunosuppressive (8-10).However, the prevalence of immune sex differences before puberty and following the onset of menopause suggests a role for non-hormonal factors as well (1,(11)(12)(13)(14).
Recently, the in uence of the XX vs. XY sex chromosome complement on immune cell activation has become increasingly appreciated.The X chromosome encodes for a large number of immune-related genes, as well as epigenetic regulators associated with lymphocyte activation and differentiation (15).
While the dosages of X-linked genes are typically balanced between males and females via the process of X chromosome inactivation (XCI) (16), X-linked genes related to immunity have been demonstrated to more readily escape XCI in immune cells than other somatic cell types, and are uniquely regulated in B lymphocytes (17)(18)(19)(20)(21). Multiple X-linked immune-related genes have been demonstrated to escape inactivation and their biallelic expression correlates with disease.For example, CD40L and CXCR3 are biallelically expressed in T cells isolated from female, but not male, systemic lupus erythematosus (SLE) patients (22,23).TLR7 and TLR8 have also been identi ed as escape genes and are overexpressed in primary B lymphocytes, monocytes, and plasmacytoid dendritic cells isolated from female SLE patients and XXY Klinefelter syndrome males (20,24).In addition to SLE, sex chromosome complementdependent in uences have been identi ed in animal models of experimental autoimmune encephalitis (EAE) (25), anti-viral immunity (26), stroke-induced neuroin ammation (27,28), and various metabolic disorders (29).
Sex has also been shown to in uence the complexity and diversity of gut microbiome populations (30)(31)(32)(33).Using a panel of over 100 diverse inbred strains of mice, Org et al. identi ed distinct gut microbial communities in male vs. female mice whose composition was in uenced by the presence of male vs. female sex hormones (31).Reciprocally, distinct sex-speci c gut microbial communities have also been demonstrated to modulate sex steroid production and in uence immune cell activation by direct and indirect mechanisms.Differences in male vs. female susceptibility to type I diabetes have been directly linked to sex-speci c gut microbiome populations capable of enhancing systemic androgen concentrations in male mice, or in female mice colonized with adoptively transferred male gut microbial communities (30,32).While these studies demonstrate that sex hormones and gut microbial communities collaborate to in uence immune activation, to our knowledge no previous study has evaluated whether sex chromosome-dependent immune phenotypes are in uenced by the gut microbiome.
Previously, our lab demonstrated that the immunomodulatory compound propanil (3,4dichloropropionanalide) enhances immune responses to heat-killed Streptococcus pneumoniae (HKSP) immunization in an XX sex chromosome complement-dependent manner using Four Core Genotype (FCG) mice (34).FCG mice exhibit one of four different genotypes: XX and XY females (ovaries) and XX and XY males (testes) and allow for the study of individual and collaborative sex hormone and sex chromosome complement-dependent effects (35)(36)(37)(38).While the mechanism mediating this propanilmediated XX-dependent immune enhancement has yet to be de ned, the contribution of circulating sex hormones was ruled out, as gonadectomy did not inhibit propanil-mediated immune enhancement (34).
In the following study, we evaluated the independent and collaborative in uences of the XX vs. XY sex chromosome complements and sex-speci c gut microbial communities on immune activation.Following immunization with HKSP, enhanced HKSP-speci c antibody secreting cells and plasma cell differentiation were noted in XX vs. XY male and female FCG mice.While Kdm6a, an X-linked epigenetic regulator with known immunomodulatory function, was demonstrated to escape X chromosome inactivation and to be overexpressed in XX-possessing cells, the ability of Kdm6a to promote plasma cell differentiation or antibody production was not identi ed to be sex chromosome-dependent.This suggested that other XX-dependent regulatory factors must be contributing to the XX-dependent phenotype.Interestingly, and importantly for the interpretation of other studies evaluating sex chromosome-dependent effects, antibiotic depletion of the endogenous microbiome reduced humoral responses to HKSP immunization in XX mice to levels similar to XY mice, but had no in uence on XY responses.This demonstrates that stronger immune responses in XX animals are microbiome-dependent.
Given that the sex chromosome complement did not alter microbiome compositions, additional studies are warranted to evaluate the mechanism by which these microbes mediate their sex chromosomedependent effect.

Materials and Methods
Four Core Genotype model

Flow cytometry
The Fc receptor of 200,000 cells were blocked with ChromPure IgG (Jackson ImmunoResearch, West Grove, PA) for 20 minutes, washed, and then stained with the following antibodies for 25 minutes on ice in the dark: rat anti-mouse B220-APC (RA3-6B2; BD Biosciences, San Diego, CA) and CD138-BV786 (281-2; BD).After staining, cells were washed and xed in 0.04% paraformaldehyde (Thermo Fisher).Live cells were determined utilizing a Live/Dead Fixable Yellow Dead Cell Stain Kit (Invitrogen, Carlsbad, CA), and where applicable absolute cell number was determined using AccuCount beads (Spherotech, Lake Forest, IL).For each sample, 10,000-30,000 cells were collected for analysis (FCS Express software) on an LSRFortessa (BD).

Gonadectomy surgeries
Bilateral castration or ovariectomy was performed on eight-to twelve-week-old mice by standard procedure (50).Brie y, mice were anesthetized with iso urane.Incisions were made through the skin and the underlying abdominal wall.The testes or ovaries were isolated and heated forceps used to cauterize the vas deferens and the blood vessel or transect the tip of the uterine horn and cauterize the blood vessels.The abdominal wall was closed with a suture and skin incisions closed with wound clips.Shamoperated mice (Sham) underwent the same procedure, but the testes or ovaries were left intact.
Gonadectomized (Gdx) and Sham mice were housed four to ve weeks following surgery before being used in experiments.
Measurement of antibody concentrations by ELISA

RNA Sequencing and analysis
After quanti cation and quality assessment of splenocyte RNA, 500ng of total RNA was used to prepare Illumina-compatible libraries using the KAPA stranded mRNA library kit (Kapa Biosystems, Wilmington, MA).Sequencing was performed as 2 x 51 cycles on an Illumina HiSeq 2000 (Marshall Genomics Core).RNA-Seq data analysis followed previously described procedures (51,52).Brie y, RNA-Seq short reads were aligned to the mm10 with subread (53).Read counts against RNA-Seq gene annotation was summarized with FeatureCounts (54).Differentially expressed genes were predicted by EdgeR with FDR less than 0.1 and a log 2 FC more than 0.585.Gene expression values (RPKM; log 2 ) across groups were visualized with GraphPad Prism version 9 for Windows (La Jolla, CA, www.graphpad.com).

qRT-PCR
RNA concentrations and purity were measured on a Nanodrop 2000 (Thermo Fisher).cDNA was synthesized with the GoScript™ Reverse Transcription kit (Promega, Madison, WI).Transcripts were ampli ed by qRT-PCR using the primers below (Life Technologies, Carlsbad, CA) and the incorporation of PowerUp™ SYBR™ Green Master Mix (Life Technologies) was measured on the StepOnePlus RT-PCR system (Applied Biosystems, Foster City, CA) to determine expression levels.The following cycling conditions were utilized: 95°C for 2 min, 40 cycles of (95°C for 15s --60°C for 1 minute), 95°C for 15s.Kdm6a expression was determined by normalization to Gapdh expression.

Western Blots
Total protein was isolated from HKSP-immunized mouse splenocytes using m-PER™ Mammalian Protein Extraction Reagent (Thermo Fisher) with 1% protease inhibitor (Cell Signaling, Danvers, MA).Protein concentrations were quanti ed using the Pierce™ Coomassie Plus (Bradford) Assay Kit (Thermo Fisher).Equal amounts of protein were boiled for 5 minutes and then resolved by SDS-Page on pre-cast Bolt 4-12% Bis-Tris Plus gels at 100V for 60-120 minutes.The gel was electrophoretically transferred to polyvinylidene uoride membranes using the iBlot™ 2 Transfer system (Invitrogen).Following transfer, membranes were blocked with 5% milk for one hour, followed by staining with primary antibody (anti-kdm6a 1:1000 and anti-β-tubulin 1:1000, Abcam, diluted in 5% milk) and incubated overnight.Membranes were then washed, incubated with secondary antibody (HRP anti-rabbit IgG) for one hour, then visualized using the SuperSignal West Pico PLUS substrate (Thermo Fisher).Blots were imaged on an iBright CL1500 (Invitrogen) imaging system and quanti ed using ImageJ with normalization to βtubulin.

RNA-FISH
Biallelic expression of Kdm6a in B cells was observed using the Stellaris® RNA FISH system (Biosearch Technologies).Brie y, B cells from HKSP-immunized mouse spleens were puri ed by negative selection with the EasySep™ mouse B cell isolation kit (STEMCELL Technologies).5x10 6 B cells were washed with PBS and resuspended in 1mL xation buffer.After 10 minutes at room temperature, cells were washed three times with 1X PBS and then permeabilized in 1mL of 70% ethanol for at least one hour at 4°C.Cells were washed and resuspended in hybridization buffer containing the Xist (mouse Xist with Quasar® 570 Dye, Biosearch Technologies) and/or Kdm6a (custom from Biosearch Technologies with Quasar® 670; sequences available upon request) probes and incubated at 37°C overnight in the dark.Cells were washed thoroughly and resuspended in 30µL of ProLong™ Glass Antifade Mountant with NucBlue™ (Invitrogen) and 5-10µl mounted on Superfrost Plus microscope slides (Thermo Fisher).2D images and Z-stacks were acquired on an inverted Nikon TI-E microscope with an A1R dual Galvano/resonant scanning confocal system equipped with four lasers (405 nm, 488 nm, 561 nm, 640 nm) and analyzed with NIS-Elements Advanced Research.For each sample, ve sections were imaged and the following quanti ed: total number of cells, number of cells with an Xist cloud, and number of cells with Xist and Kdm6a colocalization.

Microbiome Assessment
Total

Microbiome depletion
Endogenous gut microbiomes were depleted using an antibiotic cocktail containing metronidazole (10 mg/ml, Sigma-Aldrich), vancomycin (10 mg/ml, Sigma-Aldrich), neomycin (20 mg/ml, Sigma-Aldrich) and ampicillin (20 mg/ml, VWR, Radnor, PA) in sterile water.The antibiotic cocktail (100 µl) was administered via oral gavage every day for 3 days and then every other day until the end of the experiment.Controls received sterile water alone.Chow was removed from all cages 4 hours prior to antibiotic or water gavage to optimize antibiotic absorption.Microbiome depletion was veri ed using the LIVE/DEAD® BacLight™ Bacterial Viability and Counting Kit (Life Technologies) and subsequent acquisition on the LSRFortessa ow cytometer (BD).For all experiments using antibiotics, mice were housed by group to eliminate crosscontamination from fecal ingestion, provided with autoclaved drinking water and irradiated chow throughout the experiment, and provided with clean autoclaved cages after each antibiotic treatment.

Culture of SCFA-Producing Bacteria
The bacterial strains Bi dobacterium longum, Clostridium symbiosum, and Lactobacillus fermentum were purchased from ATCC.All strains were cultured in brain heart infusion (BHI) medium (Sigma-Aldrich).L. fermentum was cultured in aerobic conditions, while B. longum and C. symbiosum were cultured anaerobically using anaerobic gas jars, EZ gas packs (BD), and pre-reduced media.Under sterile conditions, bacteria were inoculated in 5-6mL culture medium and incubated at 37°C for two days.Secondary inoculations were done inoculating 1mL of the initial culture into 5-6mL of fresh culture medium.Cultures were then allowed to incubate at 37°C for 1-2 additional days.Once OD values reached at least 0.8, as measured on the xMark™ Microplate spectrophotometer (Bio-Rad), serial dilutions were made and their ODs measured.Dilutions were then plated on pre-reduced Brucella Agar with 5% sheep blood plates (B.longum and C. symbiosum; Anaerobe Systems, Morgan Hill, CA) or Blood Agar (L.fermentum; TSA with sheep blood, Remel, Lenexa, KS) plates and incubated overnight at 37°C.CFUs were counted and growth curves generated to establish standard curves for each bacterial species.Bacteria were then frozen in pre-reduced glycerol and stored at -80°C for future use.Fresh cultures were initiated from frozen stocks 6 days prior to the rst day they were needed in an experiment, with new inoculations into fresh media every other day.DNA was isolated from individual bacterial colonies and ampli ed by PCR using 16s primers (Euro ns Genomics, Louisville, KY).Sequencing of ampli ed PCR products allowed for comparison of sequences to known BLAST database for species con rmation.16s primer sequences were as follows: Forward: 5'-CGG TTA CCT TGT TAC GAC TT-3'.Reverse: 5'-AGA GTT TGA TCC TGG CTC AG-3'.

Reconstitution of SCFA-Producing Bacteria and Inulin Administration
The endogenous microbiome was depleted in all mice by antibiotic gavage as described above, with mice receiving antibiotics daily for 3 days.To reconstitute the microbiome with SCFA-producing bacteria, mice received a cocktail containing the following bacteria: B. longum (1x10 7 ), C. symbiosum (5x10 6 ), and L. fermentum (1x10 9 ) via oral gavage on Days 4 and 5. Bacterial counts were determined by OD measurements at 600nm and previously established standard growth curves.Control mice received oral gavage of sterile medium alone.Inulin (MilliporeSigma) was diluted in sterile water and provided as a second oral gavage (10mg in 100µl) on Days 4 and 5. Mice not receiving inulin were provided a second oral gavage of sterile water (100µl) alone.All mice were then immunized (i.p.) with 2x10 8 CFU HKSP on day 6.Mice receiving inulin alone continued to receive antibiotics in sterile water gavage every other day throughout the experiment, while the experimental groups received oral gavage of sterile water alone.Fecal pellets were collected at Day 0 (pre-antibiotics), Day 4 (post-antibiotics and pre-bacteria +/-inulin gavage), Day 6 (post-bacteria +/-inulin, pre-HKSP immunization), and Day 13 (euthanasia) and assessed for gut colonization status by the LIVE/DEAD® BacLight™ Bacterial Viability and Counting Kit (Life Technologies).SCFA levels were assessed by LC-MS/MS (Metabolon, Morrisville, NC).

Statistics
Statistical analyses were performed in GraphPad Prism (San Diego, CA) and QIIME2 (56, 57).Data are represented as the mean +/-SEM with each data point representing one mouse and statistical signi cance set as p < 0.05.For these studies, we employed a variety of statistical tests, including unpaired t-test, one-way ANOVA, or two-way ANOVA.Unpaired t-tests were utilized when comparing two independent groups (e.g., XXF vs. XYF females' number of antibody-secreting cells in Fig. 1A, where chromosome complement is the only distinguishing factor).One-way ANOVA followed by Tukey's multiple comparisons test was utilized when comparing data across all four genotypes (e.g., Kdm6a expression in Fig. 2A) to allow for equal consideration of chromosome complement and gonadal sex.
One-way ANOVA was followed by Dunnett's multiple comparisons test in instances where one group serves as a reference for comparison against the other groups (e.g., % CD138 + cells "Stim" as the reference group compared with increasing concentrations of GSK J4 or GSK J5 in Fig. 3B-E).Two-way ANOVA was performed to allow for the simultaneous consideration of two independent variables while accounting for the main effects of both independent variables and their interaction.Two-way ANOVA was followed by Sidak's multiple comparisons test (e.g., Fig. 1C), or Tukey's multiple comparisons test (e.g., Fig. 6D) as needed, to allow for assessment of differences among multiple conditions.Statistical test used for each analysis is denoted in the gure legends.Statistical analyses of the gut microbiome bacteria alpha and beta diversities were performed in QIIME2 and utilized Kruskal-Wallis test and permutational multivariate analysis of variance, respectively.

RESULTS
The sex chromosome complement in uences humoral responses to HKSP immunization.To evaluate whether XX vs. XY sex chromosome complements differentially regulate sexually dimorphic humoral immune responses to HKSP immunization, the FCG mouse model was utilized (Suppl.Figure 1; (35,36,61)).Ovary-bearing females with XX (XXF) or XY (XYF) sex chromosomes and testes-bearing males with XX (XXM) or XY (XYM) sex chromosomes were immunized with HKSP.One-week post-immunization, the numbers of HKSP-speci c IgM-antibody secreting cells (ASC) were assessed.Female and male FCG mice possessing an XX sex chromosome complement generated signi cantly greater numbers of HKSPspeci c IgM ASC in response to immunization compared to XY mice of the same gonadal sex (Fig. 1A & D; female XXF vs. XYF p = 0.0267; male XXM vs. XYM p = 0.0272).XXF female mice also exhibited increased percentages of CD138 + plasma cells when compared with XYF female mice (Fig. 1B; p = 0.0050).Despite not reaching statistical signi cance, CD138 + plasma cell frequencies trended similarly in XXM vs. XYM males (Fig. 1E; p = 0.0787).

Consistent with previously published reports demonstrating enhanced immune responses against
Streptococcus pneumoniae in females vs. males (62-64), a main effect of gonadal phenotype was identi ed with FCG females producing signi cantly higher numbers of HKSP-speci c IgM ASC (mean of 567.2 ± 54-1317) in comparison to gonadal FCG males (mean of 101.7 ± 13-191; unpaired t-test p < 0.0001; statistical comparison not shown in Fig. 1).To examine the role of gonadal hormones in the identi ed XX-dependent phenotype, plasma cell frequencies were evaluated in female and male shamoperated and gonadectomized (Gdx) mice immunized with HKSP.Higher percentages of CD138 + plasma cells were observed in both intact and ovariectomized XXF vs. XYF female FCG mice (Fig. 1C; XXF vs. XYF sham p = 0.0039; XXF vs. XYF gonadectomized p = 0.0003).Males trended in a similar fashion, with greater plasma cell frequencies being observed in XXM vs. XYM mice of both sham-operated and Gdx animals (Fig. 1F).Taken together, these results demonstrate that the sex chromosome complement functions independent of circulating sex hormones to modulate immune activation in response to HKSP immunization.

Identi cation of Kdm6a as a differentially expressed gene that escapes XCI
Given that HKSP-speci c immune responses were enhanced in XX vs. XY mice, we hypothesized that Xlinked gene dosage effects may be important.To test this hypothesis, RNA-Seq was performed on splenocytes isolated from HKSP-immunized male and female FCG mice.As anticipated, Xist and Sry were identi ed as genes overexpressed in XX vs. XY cells and male vs. female cells, respectively (Suppl. Figure 3).Only two additional X-linked genes were demonstrated to be overexpressed in XX vs. XY splenocytes isolated from FCG mice immunized with HKSP (threshold of log2FC > 0.585 and FDR < 0.1): Eif2s3x, and Kdm6a.KDM6a (Lysine (K)-speci c demethylase 6A, aka UTX) is a histone demethylase whose epigenetic regulatory function has previously been demonstrated to modulate other immune cells in an XX-dependent manner (65, 66), making it an interesting candidate gene for our studies.The increased expression of Kdm6a in XX vs. XY splenocytes (Fig. 2A; XXF vs. XYF females p < 0.0001; XXM vs. XYM males p = 0.0214) was con rmed by qRT-PCR in both total splenocytes and B cells isolated from FCG mice (Fig. 2B), and higher KDM6a protein levels (Fig. 2C-D) were detected in XX vs. XY B cells via western blot (main effect XX vs. XY sex chromosome; p = 0.0037, unpaired t-test).
Increased expression of an X-linked gene could indicate either biallelic expression via escape from X chromosome inactivation or increased expression from the active X chromosome.To demonstrate Kdm6a's ability to escape X chromosome inactivation, RNA-FISH was performed on B cells isolated from XXF female FCG mice one-week post-HKSP immunization.The inactive X chromosome was detected using a uorescent probe targeting Xist, a long, non-coding RNA that coats the inactive X chromosome resulting in its inactivation and formation of an Xist cloud (67, 68).Colocalization of Xist and Kdm6aspeci c probes were considered indicative of Kdm6a being expressed from the inactive X chromosome.Approximately 13% of B cells presented with an Xist cloud (mean = 13%, range of 5-29%) suggesting that they were activated in response to HKSP immunization, as naïve B cells have been shown to lack an Xist cloud (17,69).Of the B cells possessing Xist clouds, 78% (mean = 78%, range of 50-100%) exhibited colocalization of Kdm6a with Xist RNA (Fig. 2E), suggesting that Kdm6a is biallelically expressed.
The differential expression of Kdm6a was also evaluated in B cells isolated from XX vs. XY shamoperated and gonadectomized male and female mice immunized with HKSP (Fig. 2F).XX-dependent enhancement of Kdm6a expression was not impacted by castration of male mice (XXM vs. XYM p = 0.0027), and similar trends were noted in ovariectomized females (XXF vs. XYF p = 0.0652), suggesting that the XX vs. XY differential expression of Kdm6a is regulated independent of circulating sex hormones.
Inhibition of KDM6a ex vivo promotes plasma cell differentiation, but not in a sex chromosomedependent manner.
We next sought to determine whether KDM6a functions to modulate plasma cell differentiation in an XXdependent manner.Splenocytes were isolated from naïve FCG mice and stimulated with IL-4 and LPS in the presence or absence of increasing concentrations of GSK J4 or its inactive isomer GSK J5.GSK J4 is a chemical inhibitor speci c for H3K27me3 demethylases, including KDM6a (70,71).In all four genotypes (XXF, XYF, XXM, and XYM), GSK J4 (2µM) enhanced CD138 + plasma cell frequencies following ex vivo stimulation (Fig. 3A-E).Demonstrating the speci city of the GSK J4-mediated effect, its inactive isomer, GSK J5, did not impact plasma cell frequencies.Additionally, concentrations of total IgM in the supernatants of stimulated cells were not impacted by KDM6a inhibition (Fig. 3F-G, Suppl.Figure 4).Taken together, these data suggest that despite Kdm6a's overexpression in XX vs. XY cells, KDM6a's demethylase activity is not directly in uencing CD138 + plasma cell differentiation or IgM secretion in a sex chromosome-dependent manner.
The gut microbiome is required for XX-speci c immune enhancement.
Sex biases in gut microbiome diversity have been reported and demonstrated to differentially in uence immune activation (30,72).Here, we evaluated whether the gut microbiome could differentially in uence immune activation in a sex chromosome complement-dependent manner.FCG mice were administered an antibiotic (Abx) cocktail containing metronidazole (10 mg/ml), vancomycin (10 mgl/ml), neomycin (20 mg/ml) and ampicillin (20 mg/ml) via oral gavage.Control animals received sterile water alone.On day 4, mice were immunized with HKSP, and on day 10, ELISPots were performed to evaluate the number of HKSP-speci c ASC (Suppl.Figure 2).Male and female XX FCG mice with intact gut microbiomes produced more HSKP-speci c IgM ASC than XY mice in response to HKSP immunization.Interestingly, antibiotic administration signi cantly reduced XX responses (control vs. Abx; females p = 0.0025; males p = 0.0003) to levels similar to those seen in XY mice, but had no impact on XY responses (Fig. 4A-B).
The composition of FCG mouse microbiomes was then characterized by 16s rRNA sequencing to determine if distinct microbiomes in XX vs. XY mice could explain this phenotype.Based upon calculated alpha diversity metrics, namely Shannon diversity indexes and Observed Taxonomic Units (OTUs), little diversity was identi ed within XXF, XYF, XXM, and XYM samples (Fig. 4C-D).Multiple beta diversity metrics were also calculated, including Jaccard, unweighted UniFrac, weighted UniFrac, and generalized UniFrac distances.While XXF female vs. XYM male microbiomes exhibited signi cant compositional differences based upon these metrics, beta diversity was found to be similar in XXM vs. XYM males or XXF vs. XYF females in 3 of the 4 metrics assessed (Table 3).Figure 4E represents the relative abundance of taxa identi ed in FCG microbiomes and emphasizes the compositional similarities between XX vs. XY animals of the same gonadal phenotype.Since compositional differences could not fully explain the microbiome-mediated enhancement of immune responses in XX mice, it was hypothesized that sex chromosome-dependent differences in concentrations of SCFAs, major metabolites of the gut microbiome, may contribute.To test this, fecal concentrations of eight distinct SCFAs, including acetate (C2), propionate (C3), and butyrate (C4), were measured using LC-MS/MS.While the concentration of individual SCFAs varied, similar concentrations were detected between XXF vs. XYF female and XXM vs. XYM male FCG mice (Fig. 5).Interestingly, male mice tended to possess higher SCFA levels than females in general.73), we thought this was an intriguing possibility.To evaluate this potential, the endogenous gut microbiomes of male and female FCG mice were depleted using antibiotics, as described earlier.Their endogenous microbiome was then reconstituted with select SCFA-producing bacteria in the presence or absence of inulin, a ber source that is metabolized into SCFAs.Control groups underwent antibiotic microbiome depletion, but no reconstitution (Suppl.Figure 6).
All mice were then immunized with HKSP, and immune responses evaluated one week later.Live/Dead ow staining of fecal content demonstrated successful depletion of the endogenous gut microbiome and successful reconstitution in mice receiving SCFA-producers following initial antibiotic administration (Fig. 6B-C, Suppl.Figure 5).XXF females administered SCFA-producing bacteria + inulin exhibited a signi cant increase in HKSP-speci c ASC when compared to XYF animals of the same treatment group (p = 0.0062), as well as in comparison to XXF and XYF microbiome-depleted controls.No increase was detected when XXF females received SCFA-producing bacteria alone.In XYF females, neither treatment (SCFA-producing bacteria + inulin or SCFA-producing bacteria alone) exhibited increased HKSP-speci c responses (Fig. 6D), suggesting that SCFAs may function in an XX-dependent manner in females.
However, increased HKSP-speci c responses in XXM vs. XYM male mice were dependent only on the administration of the SCFA-producing bacteria, not the presence (p = 0.0205) or absence (p = 0.0025) of inulin.Similar to females, neither treatment increased responses in XY males above that of the microbiome-depleted controls (Fig. 6E).
To further evaluate if SCFAs enhance B cell function in a sex-chromosome-dependent manner, splenocytes were isolated from FCG mice and stimulated ex vivo with LPS + IL-4 in the presence or absence of increasing, biologically relevant propionate (C3) concentrations.While propionate did not impact female cell viability, high concentrations (2mM) did reduce the viability of male splenocytes after 3 days of culture (Fig. 7A-B).Plasma cell differentiation was reduced in response to propionate treatment, indicated by a dose-dependent decrease of CD138 + cell numbers, as was a dose-dependent reduction in IgM secretion regardless of gonadal sex or sex chromosome complement (Fig. 7C-F).

Discussion
In the present study, humoral immune responses against HKSP immunization were found to be differentially regulated by the presence of an XX vs. XY sex chromosome complement (Fig. 1).While the dosages of X-linked genes are typically balanced between males and females via the process of XCI (16), X-linked genes related to immunity have been demonstrated to more readily escape XCI in immune cells than other somatic cell types, and are uniquely regulated in B lymphocytes (17)(18)(19)(20)(21). Furthermore, the biallelic expression of multiple X-linked, immune-related genes has recently been reported to promote lymphocyte activation (15,16,65,66).We therefore hypothesized that X-linked gene dosage effects may contribute to the sex chromosome-dependent phenotype we identi ed in response to HKSP immunization.RNA-Seq performed on splenocytes isolated from male and female FCG mice identi ed three X-linked genes, Xist, Eif2s3x, and Kdm6a, as being overexpressed in an XX vs. XY-dependent manner (Fig. 2A, Suppl.Figure 3).The number of potential escape genes identi ed was lower than anticipated given the unique regulation of XCI in B cells but was consistent with previous reports suggesting that only 3% of X-linked genes escape inactivation in mouse embryonic broblasts.Higher percentages, upward of 15-20%, are believed to be biallelically expressed in human cells (18, 74).While Eif2s3x is a commonly recognized escape gene, Kdm6a has only recently gained attention as an epigenetic modulator of sex-biased immune activation.Functioning as a histone H3 demethylase, Kdm6a has been linked to enhanced activation of female CD4 + T cell in EAE (66), as well as female microglial activation in ischemic stroke (65).Although Kdm6a mutations have been associated with B cell cancers (75, 76), its impact on humoral responses to infection and vaccination are only beginning to be assessed.
Conservation of Kdm6a overexpression in XX vs. XY immune cells across species (mouse and human) (77) and its epigenetic regulatory function made it an interesting target for additional studies attempting to delineate the mechanisms that contribute to more robust humoral responses in XX vs. XY mice.
Following RNA-Seq, subsequent experiments con rmed Kdm6a overexpression in XX vs. XY B cells at both the RNA and protein level (Fig. 2).Since overexpression does not necessarily equate to biallelic expression, RNA-FISH was utilized to con rm Kdm6a expression on the inactive X chromosome.In these experiments, only 13% of the B cells isolated from HKSP immunized mice possessed detectable Xist clouds (data not shown), an indicator of X inactivation.Since Xist expression is low in naïve B cells and increases in response to both immunization in vivo or ex vivo stimulation (data not shown and (17, 69)), it was hypothesized that the B cells with detectable Xist clouds were re ective of activated B cell subsets (Fig. 2E).Of the B cells possessing Xist clouds, 78% demonstrated colocalization of Kdm6a and Xist signals suggesting that, similar to previous studies in mouse embryonic broblasts (77), Kdm6a is biallelically expressed by activated B cells (Fig. 2E).Due to the lack of Xist expression in naïve B cells, no conclusion could be drawn about whether Kdm6a is also biallelically expressed in this B cell subset in the current studies.However, higher Kdm6a gene expression levels were detected in XX vs. XY B cells regardless of their activation status (data not shown), suggesting that the biallelic expression pattern may also be retained in this subset.Small nucleotide polymorphisms (SNPs) present in the maternal vs.
paternal Kdm6a could be utilized to con rm Kdm6a biallelic expression in both naïve and activated B cell subsets.However, a different background strain would be needed, as C56BL6/J mice possessed no SNPs in their Kdm6a genes capable of distinguishing the maternal vs. paternal X chromosome.
Kdm6a overexpression has been demonstrated to promote T cell (19) and NK (78) function in a sexdependent manner.In B cell cancers, it has been identi ed as a tumor suppressor (79) and, in recent studies, B cell activation, isotype switching, and plasma cell differentiation were restrained by KDM6a activity (80, 81), suggesting different functional roles in different lymphocyte populations.Adding to the existing B cell data, inhibition of KDM6a activity using GSK J4 increased plasma cell differentiation in all four genotypes (XXF, XYF, XXM, XYM) at high doses following ex vivo stimulation (Fig. 3).Furthermore, KDM6a-mediated suppression of plasma cell differentiation was not sex chromosome-dependent, as XX and XY cells exhibited similar sensitivities to GSK J4-mediated inhibition.It should be noted that the inhibitory function of GSK J4 is not limited to KDM6a alone, but also impacts all JMJD3 histone demethylases.However, other JMJD3 histone methylase family members, including Kdm5c, were expressed at equivalent levels in XX and XY FCG mouse B cells (data not shown).It is possible that KDM6a in uences B cell activation and differentiation in an XX-dependent manner independent of its enzymatic activity.However, this was not assessed in the current study.
A number of additional genetic factors beyond X-linked immune gene dosage effects could be contributing to the XX-dependent phenotypes described in response to HKSP immunization.Among these are genes encoded by the Y chromosome that are not expressed in XX mice (82), X gene parental imprinting, as well as the expression of X-linked microRNAs (miRNAs) (83, 84).Going beyond these traditionally studied genetic and epigenetic regulators, we propose that additional factors, including the gut microbiome, may also be in uencing immune responses in an XX-dependent manner.
The gut microbiome has more recently been established as an important regulator of immunity and has been demonstrated to differentially modulate immune activation in males and females (30)(31)(32)85).To our knowledge, the collaborative role of the gut microbiome and XX sex chromosome complement has not been evaluated.Antibiotic depletion of the gut microbiome reduced humoral responses in XX mice to levels similar to that of XY, while having no impact on the magnitude of XY responses (Fig. 4A-B), suggesting a synergistic effect.Consistent with previous reports (86-88), sex-speci c differences were noted in the composition of male vs. female gut microbiomes (Fig. 4C-E).However, minimal differences were identi ed in the microbiome compositions of XXF vs. XYF females or XXM vs. XYM males, or in the levels of SCFAs between XX vs. XY mice of the same gonadal sex (Fig. 5).This led us to consider other mechanisms by which the gut microbiome could modulate immune responses in an XX-dependent manner.Our laboratory previously demonstrated that the immunomodulatory compound propanil enhances responses to HKSP immunization in an XX-dependent manner (34).Given that a major metabolite of propanil is also a metabolite of the gut microbiome, namely propionate, we hypothesized that propionate may likewise be capable of in uencing humoral immune responses in an XX-dependent manner.In lymphocytes, the immunomodulatory effects of SCFAs have most commonly been attributed to their histone deacetylase (Class I/II) inhibitory function (40,42,(89)(90)(91)(92).In B cells, propionate has previously been demonstrated to inhibit HDAC activity, resulting in histone H3 hyper-acetylation, plasma cell differentiation, and class-switching (40).Subsequent opposing studies suggested that SCFAs both promote and suppress B cell responses, depending upon the exposure dose (40,93).To our knowledge, no previous report has evaluated whether SCFAs in uence immune responses differently in males vs. females or if these effects may be sex chromosome-dependent.
Here we attempted to address whether SCFAs in uence humoral immunity in an XX-dependent manner in two ways.This rst involved antibiotic depletion of gut bacteria and reconstitution of the microbiome with SCFA-producing species in the presence or absence of a ber source that promotes SCFA production prior to HKSP immunization.While enhancement of XX-dependent responses required the presence of gut microbiome bacteria, this enhancement appeared to be SCFA-dependent in females only and not in males (Fig. 6D-E).Interestingly, reconstitution of the microbiomes with SCFA-producing bacteria seemed more e cient in female vs. male cells (Fig. 6B-C).These differences in reconstitution, along with differing circulating sex hormones, may explain the observed differences in females vs. males' dependence on SCFA production.Interestingly, the main effect of gonadal sex, in which females responded more robustly to HKSP immunization than males (Fig. 1), was lost in mice possessing only select SCFA-producing bacteria.This suggests that sex-speci c gut microbiome compositions in uence the robustness of an immune response.However, those compositional differences alone are not mediating the XX sex Taken together, these data suggest that the gut microbiome in uences immune responses in a multifaceted manner, with contributions from SCFA production as well as other, not yet de ned microbial components.Discrepancies between in vivo and ex vivo results may also be re ective of additional sexspeci c collaborations occurring in our in vivo studies, which could implicate the organizational in uence of sex hormones on the gut-microbiome mediated effects.
While our study provides valuable insights into the complex interactions between the sex chromosome complement and gut microbiome in shaping immune responses, it is essential to acknowledge the limitations of the FCG mouse model.Others have demonstrated similar levels of circulating sex hormones in XX vs. XY mice of the same gonadal sex (28, 94-96), but variations in gonadal morphology and function between XX and XY mice of the same gonadal sex, as well as potential differences in the phenotypic responses to cyclic ovarian hormones (97,98), should be considered when interpreting our ndings.We attempted to control for these variables by gonadectomy when possible, but the potential for hormone organizational effects prior to gonadectomy cannot be overlooked.Additionally, the variability in gut microbiome composition observed between animal facilities is a challenge whenever studying its role in biological processes.We attempted to mitigate this challenge by depleting the endogenous microbiome and reconstituting with select species, which may be a valuable strategy for obtaining reproducible results in multiple settings.

Perspectives and Signi cance
While the impacts of sex hormones, sex chromosome complement, and the gut microbiome on immunity have been well characterized (1,37,(99)(100)(101), the present study underscores the essential consideration that these three biological systems are intrinsically interconnected.The collaboration between sex hormones and sex chromosomes has previously been evaluated, but less is known about the interplay between these sex-speci c factors and the gut microbiome.Sex hormones are known to be crucial regulators of microbiome colonization (30,31,85), and conversely, the gut microbiota can in uence hormone production and bioactivity (32).Most previous studies investigating microbiomedependent in uences have focused on identifying sex-speci c microbiome populations to explain dimorphic responses.However, in the current studies, while attempting to delineate the underlying mechanisms contributing to sex biases in immune responses to HKSP immunization, we demonstrated that similar gut microbiomes can in uence immune sex biases in an XX sex chromosome-dependent manner.

Declarations
Ethics approval and consent to participate Studies were conducted in accordance with all federal and institutional guidelines for animal use and were approved by the WVU Institutional Animal Care and Use Committee, protocol #1603001079.

Figure 4 Similar
Figure 4

Table 1
The genotype of the offspring was determined by PCR ampli cation of the following genes: Sry, Ymt, and Myo, using DNA isolated from tail samples or ear punches obtained at weaning.QIAGEN Fast Cycling PCR kit (Qiagen, Louisville, KY) was used for PCR ampli cation.Sry, Ymt, and Myo primer sequences (Invitrogen): − Sry) males with C57BL/6J females (The Jackson Laboratory).All purchased animals were allowed to acclimate for one week prior to use.The sex determining region of the Y chromosome, the Sry gene, had previously been deleted from the Y chromosome of XY − Sry mice and inserted as a transgene onto autosome 3. Breeding of XY − Sry male mice with wildtype C57BL/6 female mice produced FCG mice: XX or XY gonadal females (XXF and XYF) and XX or XY gonadal males (XXM and XYM), as shown in Supplemental Fig.1.FCG mice were weaned at 21 days of age.
12 hr light-dark cycle with food and water provided ad libitum.Studies were conducted in accordance with all federal and institutional guidelines for animal use and were approved by the WVU Institutional Animal Care and Use Immulon 2 plates (ThermoLabsystems, Pittsburgh, PA) were coated overnight at 4°C with goat antimouse human adsorbed unlabeled IgM (Southern Biotech; 100 µl/well).Plates were washed, blocked with 3% BSA in PBS at 37°C overnight, washed, and 100µl/well of four two-fold dilutions of sera in PBS + 1% BSA were added starting at 1:2.Sample containing plates were incubated for 1 hour at 37°C and washed.Goat anti-mouse AP conjugated antibodies (Southern Biotech; 100µl/well) were added for 1 hour at 37°C.Plates were washed and 100µl of phosphatase substrate tablets (Sigma-Aldrich) dissolved in p-Nitrophenyl Phosphate, Disodium Salt (PNPP) buffer was added to wells.Absorbance was read at 405nm on an xMark™ Microplate Spectrophotometer with the Microplate Manager™ Software (Bio-Rad, Hercules, CA).Standard curves were generated using serial dilutions of puri ed rat anti-mouse IgM (Clone II/41, BD), and the 4-parameter t equation used to calculate sample concentrations.
(55)was extracted from fecal samples using the DNeasy PowerSoil DNA isolation kit (Qiagen) according to the manufacturer's recommended protocol.PCR ampli cation of the V3 region of the 16sRNA RNA gene was performed by utilizing high pressure liquid chromatography-puri ed primers (Integrated DNA Technologies; Coralville, IA), AccuPrime PCR Kit (Invitrogen) and cycling conditions previously described by Fadrosh et al.(55).Brie y, cycling conditions included: 95°C for 6 minutes denature; 95°C for 2 minutes, 50°C for 2 minutes, 72°C for 2 minutes 30 cycles; 72°C for 4 minutes extend.Each reaction contained 0.5 µl Taq polymerase, 5 µl 10x buffer 1(600 mM Tris-SO4 (pH 8.9), 180 mM (NH4)2SO4, 20 mM MgSO4, 2 mM dGTP, 2 mM dATP, 2 mM dTTP, 2 mM dCTP, thermostable AccuPrime™ protein, 10% glycerol), 20 µM forward primer, 20 µM reverse primer, and up to 60 ng DNA in a total volume of 50 µl.Primer sequences are available upon request.Following quantitation and quality (59)rol analysis of the ampli ed 16s rRNA ampli cation product, paired-end sequencing (2 x 150 bp) was performed using the Illumina MiSeq located in the Genomics Core Facility at WVU. Microbiome sequencing les were analyzed using QIIME2 (version 2020.11)(56,57).Sequencing quality was inspected using fastQC (58).DADA2(59)was used to optimize the parameter for quality control and read trimming.Taxonomy assignments were performed using the SILVA 132 (60) database at 97% identities.Rarefaction curve analysis on alpha diversity was used to estimate the sampling completeness and for OTU calculations.Beta diversity metrics calculated included Jaccard distances, unweighted UniFrac distances, weighted UniFrac distances, and generalized UniFrac distances.Signi cance in the difference between alpha and beta diversities was based on Kruskal-Wallis test and permutational multivariate analysis of variance, respectively.

Table 3
*p ≤ 0.05; comparison of index distances using QIIME2 plugins using PERMANOVA Despite no detected differences in XX vs. XY SCFA concentrations, it was not known if SCFAs function differently in XX vs. XY mice.Given our previous studies demonstrating that propanil enhances HKSP immune responses in an XX-dependent manner ( chromosome-dependent effects noted following HKSP immunization, and thus requires additional study.Additionally, we tested the in uence of SCFAs on XX vs. XY B cell activation in response to ex vivo mitogen stimulation.Using concentrations similar to those previously reported by Kim et al. and Sanchez et al., a reduction in viability was noted only at high propionate concentrations in male cells.Despite this, propionate was demonstrated to decrease mitogen-induced plasma cell differentiation in a dosedependent manner, in concurrence with Sanchez et al., and it does so similarly in all four genotypes.