Gut Microbial Membership Modulates CD4 T Cell Reconstitution and Function after Sepsis

Abstract

Transient lymphopenia is one hallmark of sepsis, and emergent data indicate the CD4 T cell compartment in sepsis survivors is numerically and functionally altered (when examined at the Ag-specific level) compared with nonseptic control subjects. Previous data from our laboratory demonstrated Ag-independent, lymphopenia-induced homeostatic proliferation to be a contributing mechanism by which CD4 T cells numerically recover in sepsis survivors. However, we reasoned it is also formally possible that some CD4 T cells respond directly to Ag expressed by gut-resident microbes released during polymicrobial sepsis. The effect of gut microbiome leakage on CD4 T cells is currently unknown. In this study, we explored the number and function of endogenous CD4 T cells specific for segmented filamentous bacterium (SFB) after cecal ligation and puncture (CLP)-induced sepsis using mice that either contained or lacked SFB as a normal gut-resident microbe. Interestingly, SFB-specific CD4 T cells underwent Ag-driven proliferation in CLP-treated SFB(+), but not in SFB(-), mice. Moreover, CLP-treated SFB(+) mice showed resistance to secondary lethal infection with recombinant SFB Ag-expressing virulent Listeria (but not wild-type virulent Listeria), suggesting the CLP-induced polymicrobial sepsis primed for a protective response by the SFB-specific CD4 T cells. Thus, our data demonstrate that the numerical recovery and functional responsiveness of Ag-specific CD4 T cells in sepsis survivors is, in part, modulated by the intestinal barrier's health discreetly defined by individual bacterial populations of the host's microbiome.

Figure 1

Naïve B6J and B6NTac mice differ in number of SFB-specific CD4 T cells and frequency of CD44^hi SFB-specific CD4 T cells. (A) Representative flow plots show the gating strategy used in tetramer-enriched cell fractions to detect Ag-specific CD4 T cell populations. Shown are examples used to detect 2W1S:I-A^b- and 3340-N5:I-A^b-specific CD4⁺ T cells. Gating for p:I-A^b-specific cells was determined using CD8⁺ T cells as an internal negative control for tetramer binding. (B) Number of 2W1S:I-A^b- and 3340-N5:I-A^b-specific CD4 T cells and (C) frequency of CD44^hi 2W1S:I-A^b- and 3340-N5:I-A^b-specific CD4 T cells in the spleens of naïve B6J and B6NTac mice. Statistical significance was determined using group-wise, one-way ANOVA with multiple-testing correction using the Holm-Sidak method, and α = 0.05. Data shown are from 9 mice/group.

Figure 2

Differential response of SFB-specific CD4 T cells in CLP-treated B6J and B6NTac mice. (A) Number of total CD4 T cells in spleens of B6J and B6NTac mice on d 2 and recovery by d 30 after CLP. (B) Representative plots show the number of SFB-specific CD4 T cells and frequency of CD44^hi SFB-specific CD4 T cells in sham- and CLP-treated B6J and B6NTac mice. (C-D) Number of SFB-specific CD4 T cells and frequency of CD44^hi SFB-specific CD4 T cells in sham- and CLP-treated B6J and B6NTac mice 2 or 30 d after surgery. Statistical significance was determined using group-wise, one-way ANOVA with multiple-testing correction using the Holm-Sidak method, and α = 0.05. Data shown represent 2 independent experiments, with 4-5 mice/group in each experiment.

Figure 3

Horizontal transfer of SFB by co-housing permits the expansion of SFB-specific CD4 T cells in B6J mice after CLP. B6J and B6NTac mice were housed separately or co-housed for 4 weeks. (A) The presence of SFB in fecal pellets in individual mice was determined by PCR prior to sham or CLP surgery. Each symbol represents an individual mouse. (B-C) The number of SFB-specific CD4 T cells and frequency of CD44^hi SFB-specific CD4 T cells in the spleens of B6J, B6NTac, and co-housed B6J mice on d 2 and 30 after CLP surgery. Statistical significance was determined using group-wise, one-way ANOVA with multiple-testing correction using the Holm-Sidak method, and α = 0.05. Data shown are the representative from at least 2 independent experiments per population analyzed, with 4-5 mice/group in each experiment.

Figure 4

Transient reduction of CD4 T cells without bacteremia is not sufficient to drive SFB-specific CD4 T cell expansion in B6NTac mice. (A) B6J and B6NTac mice were injected with 25 μg of the anti-CD4 depleting mAb GK1.5 or an isotype control mAb. Mice were bled before mAb injection and on d 3 and 33 after injection. (B) The frequency of CD45.2⁺CD3⁺CD4⁺ T cells in the blood was determined. (C) On d 33, the number of SFB-specific CD4 T cells in the spleens was determined by tetramer enrichment and flow cytometry. (D) B6J and B6NTac mice were injected with 10 mg/kg LPS or PBS. (E) Spleens were collected on d3 and 30 to measure the frequency of CD45.2⁺CD3⁺CD4⁺ T cells. (F) On d 30, the number of SFB-specific CD4 T cells in the spleens was determined by tetramer enrichment and flow cytometry.

Figure 5

SFB colonization changes the function of SFB-specific CD4 T cells in sepsis survivors. (A-B) B6J and B6NTac mice were infected with att. Lm-SFB (10⁷ CFU in 0.1 ml i.v.) 30 d after sham or CLP surgery. (A) Proliferative capacity – The number of LLO₁₉₀- and SFB-specific CD4 T cells was determined in the spleen 7 d after infection. (B) Functional capacity – 7 d after infection, splenocytes were stimulated for 4 h with PMA/ionomycin. The samples were enriched for SFB-specific CD4 T cells and IFNγ production was determined. Representative flow plots show the frequency of SFB-specific CD4 T cells producing IFNγ. The frequency and number of IFNγ⁺ SFB-specific CD4 T cells are graphed. Statistical significance in A-B was determined using group-wise, one-way ANOVA analyses followed by multiple-testing correction using the Holm-Sidak method, with α = 0.05. Data shown represent 2 independent experiments, with 3-5 mice/group in each experiment.

Figure 6

SFB colonization influences the protective capacity of SFB-specific CD4 T cells in sepsis survivors to secondary infection. (A-B) B6J and B6NTac mice were infected with vir. Lm-SFB (5 × 10³ CFU in 0.1 ml i.v.) 30 d after sham or CLP surgery. (A) Mice were sacrificed on d 5 post-infection to determine Listeria burden in the liver, while other cohorts (B) were monitored for survival. (C) Additional cohorts of B6J mice co-housed with B6NTac mice for 4 weeks before sham or CLP surgery were similarly infected with vir. Lm-SFB 30 d after surgery. (D) B6NTac mice were infected with vir. wild-type Lm (Lm-WT) or Lm-SFB (5 × 10³ CFU in 0.1 ml i.v.) 30 d after sham or CLP surgery. Some of the CLP-treated mice were depleted of CD4 T cells using the GK1.5 mAb (3 daily injection of 100 μg i.p.) immediately before infection. Survival was monitored. Statistics shown were obtained using Mann-Whitney U test statistics (for B) and one-way ANOVA analyses (for A, C, and D) with multiple-testing correction using the Holm-Sidak method, and α = 0.05. Survival data are cumulative from 3 independent experiments with 5 mice/group in each experiment.