Morphine-induced intestinal microbial dysbiosis drives TLR-dependent IgA targeting of gram-positive bacteria and upregulation of CD11b and TLR2 on a sub-population of IgA+ B cells

Abstract

IgA binding dictates the composition of the intestinal microbiome and reflects dysbiotic states during chronic disease. Both pathogenic and commensal bacteria differentially bind to IgA with varying outcomes. Little is known regarding IgA dynamics immediately following microbial dysbiosis. Recent work shows that morphine treatment rapidly induces microbial dysbiosis within hours of administration. This microbial shift is characterized by the expansion of pathogenic bacteria with a concurrent decrease in commensal bacteria. Because of this rapid microbial shift, a murine model of chronic morphine treatment was used to gain insight on the host IgA response during early microbial disruption. Within 24 h, morphine treatment induces microbial dysbiosis which disrupts IgA-bacterial homeostasis, resulting in an increased concentration of unbound IgA with a corresponding decrease in the frequency of IgA-bound bacteria. Additionally, the increased concentration of unbound IgA is dependent on the microbiome, as microbial depletion abolishes the increase. At 48 h of morphine treatment, the frequency of IgA-bound bacteria increases and IgA-seq reveals increased IgA targeting of gram-positive bacteria. Both a whole-body TLR2 KO and treatment with the TLR inhibitor OxPAPC resulted in abrogation of IgA binding to bacteria, implicating modulation of IgA binding through TLR signaling. Finally, we identify that a sub-population of IgA⁺ B cells in the intestinal lamina propria has increased CD11b and TLR2 expression at 24 h of morphine treatment which could be a potential source of the observed IgA that targets gram-positive bacteria. Together, we demonstrate for the first time the role of TLR2 in IgA targeting of intestinal bacteria, and this study sheds light on the IgA dynamics during the initial hours of microbial dysbiosis.

Keywords: IgA; IgA+CD11b+ cells; IgA-seq; TLR2; immunoglobulin a; intestinal immunity; microbiome; morphine; opioids; small intestine.

Figure 1.

Morphine treatment induces intestinal microbial dysbiosis accompanied by increased concentration of unbound IgA. (a). 16s rRNA sequencing was performed on bacteria isolated from ileal luminal content from mice sacrificed after 24 h of continuous morphine treatment (n = 10-11). Principal coordinate analysis (PCoA) plots with Bray-Curtis distance were generated from 16s rRNA sequencing. (b). FlowSoFine and vegan R packages were used to generate a PCoA plot (left panel) with Bray-Curtis distance from FCS files of bacterial flow cytometry. Representative scatter plots of individual samples from each group (right panels) from FlowSoFine. (c-e). Unbound IgA concentration was measured via ELISA from luminal content taken from duodenum, jejunum, or ileum after 24 h of 25 mg morphine or placebo treatment (n = 4-6). Symbols represent individual mice. Data points are pooled from 2–3 independent experiments. Adjusted p-values were displayed with Benjamini-Hochberg correction method (a and b). *p < 0.05 **p < 0.01 ***p < 0.001 ****p < 0.0001 using Mann–Whitney U test (c, and d) or two-way analysis of variance with Tukey correction (e).

Figure 2.

An intact microbiome is required for the morphine-induced increase in ileum IgA concentration. (a). IgA ELISA of ileal luminal content from microbiome-depleted or vehicle-treated mice following 24 h of continuous morphine treatment (n = 8-10). (b). PCoA plots of 16s rRNA sequencing from ileal luminal content taken from WT and RAG KO mice 24 h after implantation with 25 mg morphine or placebo pellets (n = 8-10). (c). Percent IgA-bound bacteria from ileal luminal content isolated from WT mice 24 h after implantation with 25 mg slow-release morphine or placebo pellet (n = 11). (d). Representative flow cytometry plots of DAPI⁺IgA⁺ events from (c). Symbols represent individual mice. Mean and standard deviation are shown. Data points are pooled from 3-4 independent experiments. *p < 0.05 **p < 0.01 ***p < 0.001 ****p < 0.0001 using two-way analysis of variance with Tukey correction (a), Benjamini–Hochberg correction method (b) and Mann–Whitney U test (c).

Figure 3.

The proportion of IgA-bound intestinal bacteria increases at 48 h of morphine treatment. (a). 16s rRNA sequencing was performed on bacteria isolated from ileal luminal content from mice sacrificed after 48 h of continuous morphine treatment (n = 8-10). PCoA plot with Bray-Curtis distance was generated from 16s rRNA sequencing. (b). FlowSoFine and vegan R packages were used to generate a PCoA plot (left panel) with Bray-Curtis distance from FCS files of bacterial flow cytometry. Representative scatter plots of individual samples from each group (right panels) from FlowSoFine. (c). Unbound IgA concentration ELISA from ileal luminal content 48 h after implantation of 25 mg morphine or placebo pellet (n = 7). (d). Percent IgA-bound bacteria from ileal luminal content isolated from WT mice 48 h after implantation with 25 mg slow-release morphine or placebo pellet (n = 9-10). (e). Representative flow cytometry plots of DAPI⁺IgA⁺ events from (d). Symbols represent individual mice. Mean and standard deviation are shown. Data points are pooled from 2-4 independent experiments. Adjusted p-values were displayed with Benjamini–Hochberg correction method (a and b). *p < 0.05 **p < 0.01 ***p < 0.001 ****p < 0.0001 using Mann–Whitney U test (c and d).

Figure 4.

IgA binding to gram-positive bacteria is increased at 48 h of morphine treatment. (a). DNA was extracted from ileal luminal content bacteria after cell sorting to obtain IgA⁺ and IgA⁻ fractions for mice implanted with 25 mg morphine or placebo pellets for 48 h (n = 9-10). 16s rRNA was sequenced for all samples and IgA indices were calculated using the Kau Index. Average IgA indices for bacteria are shown (red bars = morphine, black bars = placebo). Plots showing bacterial taxa with (b) increased or (c) decreased IgA indices. Relative abundance of selected bacteria from pre-sort sequences of (d) placebo- or (e) morphine-treated mice. Symbols represent individual mice. Mean and standard deviation are shown. Data points are pooled from three independent experiments. *p < 0.05 **p < 0.01 ***p < 0.001 ****p < 0.0001 using unpaired T test (a-c).

Figure 5.

TLR2 is required for the morphine-induced increase in the frequency of IgA-bound bacteria. (a). Percent IgA-bound bacteria from ileal luminal content bacteria of WT and TLR2 KO mice implanted with 25 mg morphine or placebo pellet for 48 h (n = 4-5). (b). Representative flow plots of IgA-bound bacteria. (c). Mice were injected intraperitoneally with 200 μg of OxPAPC or 0.9% saline 24 h after implantation with a 25 mg morphine or placebo pellet before sacrifice at 48 h. (d). Percent IgA-bound bacteria in WT mice implanted with a 25 mg morphine or placebo pellet mice following saline or OxPAPC injection (n = 4-10). (e). Representative flow cytometry plots of IgA-bound bacteria. Symbols represent individual mice. Mean and standard deviation are shown. Data points are pooled from three independent experiments. *p < 0.05 **p < 0.01 ***p < 0.001 ****p < 0.0001 using two-way analysis of variance with Tukey correction (a and d).

Figure 6.

IgA⁺ immune cells in the lamina propria upregulate CD11b and TLR2 following morphine treatment (a-f). Flow cytometry on ileum lamina propria immune cells isolated 24 h after implantation with placebo or 25 mg morphine pellets (n = 5). (a). Representative flow cytometry plots of intestinal IgA and CD11b expression on immune cells. (b). Percentage of IgA⁺CD11b⁺ cells. (c). Absolute counts of IgA⁺CD11b⁺ cells. (d). Absolute counts of IgA⁺CD11b⁻ cells. (e) Representative flow cytometry plots of TLR2 expression on IgA⁺CD11b⁺ immune cells. (f). Percentage of IgA⁺CD11b⁺TLR2⁺ cells. (g). Absolute count of IgA⁺CD11b⁺TLR2⁺ cells. Symbols represent individual mice. Mean and standard deviation are shown. Data points are pooled from three independent experiments. *p < 0.05 **p < 0.01 ***p < 0.001 ****p < 0.0001 using two-way analysis of variance with Tukey correction (b, c, d, f, and g).

Figure 7.

Morphine-induced microbial dysbiosis disrupts IgA-bacterial homeostasis which precedes TLR-dependent IgA targeting of intestinal bacteria.