Dataset Information

Quantifying HIV-1-Mediated Gut CD4⁺ T Cell Deathin the Lamina Propria Aggregate Culture (LPAC) Model.

ABSTRACT: Gut CD4 T cells are major targets of HIV-1 and are massively depleted early during infection. To better understand the mechanisms governing HIV-1-mediated CD4 T cell death, we developed the physiologically-relevant Lamina Propria Aggregate Culture (LPAC) model. The LPAC model is ideal for studying CD4 T cell death induced by clinically-relevant Transmitted/Founder (TF) HIV-1 strains and is also suitable for studying how enteric microbes and soluble factors (e.g., Type I Interferons) impact LP CD4 T cell death and function. Here, we detail the protocol to establish LP CD4 T cell infection using a process of spinoculation, the subsequent evaluation of infection levels using multicolor flow cytometry and the determination of overall LP CD4 T cell death using absolute LP CD4 T cell counts. We also describe the preparation of virus stocks of Transmitted/Founder (TF) HIV-1 infectious molecular clones that were successfully used in the LPAC model.

SUBMITTER: Dillon SM

PROVIDER: S-EPMC7842360 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Publications

Quantifying HIV-1-Mediated Gut CD4<sup>+</sup> T Cell Deathin the Lamina Propria Aggregate Culture (LPAC) Model.

Dillon Stephanie M SM Guo Kejun K Castleman Moriah J MJ Santiago Mario L ML Wilson Cara C CC

Bio-protocol 20200120 2

Gut CD4 T cells are major targets of HIV-1 and are massively depleted early during infection. To better understand the mechanisms governing HIV-1-mediated CD4 T cell death, we developed the physiologically-relevant Lamina Propria Aggregate Culture (LPAC) model. The LPAC model is ideal for studying CD4 T cell death induced by clinically-relevant Transmitted/Founder (TF) HIV-1 strains and is also suitable for studying how enteric microbes and soluble factors (<i>e.g.</i>, Type I Interferons) impac ...[more]

PMID: 33654719

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Project description:An important function of the gut microbiome is the fermentation of non-digestible dietary fibers into short chain fatty acids (SCFAs). The three primary SCFAs: acetate, propionate, and butyrate, are key mediators of metabolism and immune cell function in the gut mucosa. We previously demonstrated that butyrate at high concentrations decreased human gut lamina propria (LP) CD4 T cell activation in response to enteric bacteria exposure in vitro. However, to date, the mechanism by which butyrate alters human gut LP CD4 T cell activation remains unknown. In this current study, we sought to better understand how exposure to SCFAs across a concentration range impacted human gut LP CD4 T cell function and activation. LP CD4 T cells were directly activated with T cell receptor (TCR) beads in vitro in the presence of a physiologic concentration range of each of the primary SCFAs. Exposure to butyrate potently inhibited CD4 T cell activation, proliferation, and cytokine (IFNγ, IL-17) production in a concentration dependent manner. Butyrate decreased the proliferation and cytokine production of T helper (Th) 1, Th17 and Th22 cells, with differences noted in the sensitivity of LP versus peripheral blood Th cells to butyrate's effects. Higher concentrations of propionate and acetate relative to butyrate were required to inhibit CD4 T cell activation and proliferation. Butyrate directly increased the acetylation of both unstimulated and TCR-stimulated CD4 T cells, and apicidin, a Class I histone deacetylase inhibitor, phenocopied butyrate's effects on CD4 T cell proliferation and activation. GPR43 agonism phenocopied butyrate's effect on CD4 T cell proliferation whereas a GPR109a agonist did not. Our findings indicate that butyrate decreases in vitro human gut LP CD4 T cell activation, proliferation, and inflammatory cytokine production more potently than other SCFAs, likely through butyrate's ability to increase histone acetylation, and potentially via signaling through GPR43. These findings have relevance in furthering our understanding of how perturbations of the gut microbiome alter local immune responses in the gut mucosa.

Project description:The RAG1/RAG2 endonuclease (RAG) initiates the V(D)J recombination reaction that assembles immunoglobulin heavy (IgH) and light (IgL) chain variable region exons from germline gene segments to generate primary antibody repertoires. IgH V(D)J assembly occurs in progenitor (pro-) B cells followed by that of IgL in precursor (pre-) B cells. Expression of IgH ? and IgL (Ig? or Ig?) chains generates IgM, which is expressed on immature B cells as the B-cell antigen-binding receptor (BCR). Rag expression can continue in immature B cells, allowing continued Ig? V(D)J recombination that replaces the initial V?J? exon with one that generates a new specificity. This 'receptor editing' process, which can also lead to Ig? V(D)J recombination and expression, provides a mechanism whereby antigen encounter at the Rag-expressing immature B-cell stage helps shape pre-immune BCR repertoires. As the major site of postnatal B-cell development, the bone marrow is the principal location of primary immunoglobulin repertoire diversification in mice. Here we report that early B-cell development also occurs within the mouse intestinal lamina propria (LP), where the associated V(D)J recombination/receptor editing processes modulate primary LP immunoglobulin repertoires. At weanling age in normally housed mice, the LP contains a population of Rag-expressing B-lineage cells that harbour intermediates indicative of ongoing V(D)J recombination and which contain cells with pro-B, pre-B and editing phenotypes. Consistent with LP-specific receptor editing, Rag-expressing LP B-lineage cells have similar VH repertoires, but significantly different V? repertoires, compared to those of Rag2-expressing bone marrow counterparts. Moreover, colonization of germ-free mice leads to an increased ratio of Ig?-expressing versus Ig?-expressing B cells specifically in the LP. We conclude that B-cell development occurs in the intestinal mucosa, where it is regulated by extracellular signals from commensal microbes that influence gut immunoglobulin repertoires.

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Dataset Information

Quantifying HIV-1-Mediated Gut CD4⁺ T Cell Deathin the Lamina Propria Aggregate Culture (LPAC) Model.

Publications

Quantifying HIV-1-Mediated Gut CD4<sup>+</sup> T Cell Deathin the Lamina Propria Aggregate Culture (LPAC) Model.

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Dataset Information

Quantifying HIV-1-Mediated Gut CD4+ T Cell Deathin the Lamina Propria Aggregate Culture (LPAC) Model.

Publications

Quantifying HIV-1-Mediated Gut CD4<sup>+</sup> T Cell Deathin the Lamina Propria Aggregate Culture (LPAC) Model.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Quantifying HIV-1-Mediated Gut CD4⁺ T Cell Deathin the Lamina Propria Aggregate Culture (LPAC) Model.