image_1_Azithromycin Clears Bordetella pertussis Infection in Mice but Also Modulates Innate and Adaptive Immune Responses and T Cell Memory.jpeg

<p>Treatment with the macrolide antibiotic azithromycin (AZM) is an important intervention for controlling infection of children with Bordetella pertussis and as a prophylaxis for preventing transmission to family members. However, antibiotics are known to have immunomodulatory effects independent of their antimicrobial activity. Here, we used a mouse model to examine the effects of AZM treatment on clearance of B. pertussis and induction of innate and adaptive immunity. We found that treatment of mice with AZM either 7 or 14 days post challenge effectively cleared the bacteria from the lungs. The numbers of innate immune cells in the lungs were significantly reduced in antibiotic-treated mice. Furthermore, AZM reduced the activation status of macrophages and dendritic cells, but only in mice treated on day 7. Early treatment with antibiotics also reduced the frequency of tissue-resident T cells and IL-17-producing cells in the lungs. To assess the immunomodulatory effects of AZM independent of its antimicrobial activity, mice were antibiotic treated during immunization with a whole cell pertussis (wP) vaccine. Protection against B. pertussis induced by immunization with wP was slightly reduced in AZM-treated mice. Antibiotic-treated wP-immunized mice had reduced numbers of lung-resident memory CD4 T cells and IL-17-production and reduced CD49d expression on splenic CD4 T cells after challenge, suggestive of impaired CD4 T cell memory. Taken together these results suggest that AZM can modulate the induction of memory CD4 T cells during B. pertussis infection, but this may in part be due to the clearance of B. pertussis and resulting loss of components that stimulate innate and adaptive immune response.</p

CD8⁺ T cell differentiation status during chronic infection.

Mixed bone marrow chimeric animals were infected with 106 PFU of MCMV and sacrificed at 90 dpi. (A) Absolute count of KLRG1+CD27- CD8+ T cells in spleen and lungs of BMC animals at 90 dpi. (B) Frequency of CM and KLRG1-CD27+ T cells among primed CD45.2+/+ CD8+ T cells (CD44+CD11a+) from mesenteric LN of chronically infected mice. (C) Quantification of CM CD8+ T cells from CD45.2+/+ compartment in blood, spleen, lungs and mesenteric LN. (D) Absolute count of CX3CR1+ CD8+ T cells in spleen and lungs of BMC animals at 90 dpi. Data are pooled from two independent experiments and each dot represent one mouse. Statistically significant differences are highlighted; *, p (TIF)</p

NFAT signaling regulates effector CD8⁺ T cell differentiation during chronic infection.

Mixed BMC animals were infected with 106 PFU of MCMV and analyzed at 90 dpi. (A) Representative flow-cytometric plots showing KLRG1+CD27- and KLRG1-CD27+populations from BMC mice with WT and NFATc1c2 DKO BM. The plots are pre-gated on primed (CD44+CD11a+) CD45.2+ CD8+ T cells (live CD3+CD8+). (B) Pairwise comparison of KLRG1+CD27- and KLRG1-CD27+CD8+ T cell frequencies in blood, spleen and lungs of individual mice during chronic MCMV infection. Lines connect data from individual animals (C) Flow-cytometric plots showing representative central memory (CM) populations among primed blood CD8+ T cells of chronically infected mice (left). Kinetics of these CM populations in blood are shown on the right Lines connect group means at indicated time points, error bars are SEM. (D) Percentage of CM CD8+ T cells in spleen and lungs at 90 dpi. Bar plots represent the group average, error bars are SEM and each dot represents mouse. (E) Percentage of CM cells among M45 and M38 tetramer specific CD8+ T cells from spleen at 90 dpi. Bar plots represent mean ± SEM and each dot is a mouse. (F) Representative flow-cytometric plots of blood CD8+ T cells showing CXCR3+ population among primed (CD44+CD11a+) cells (left). Mean CXCR3+ CD8+ T cells population from blood, spleen and mesenteric LN at 90 dpi are shown as mean ± SEM, each dot is a mouse. (G) Representative flow-cytometric plots of blood CD8+ T cells showing CX3CR1+ population among primed (CD44+CD11a+) cells (left). Mean CX3CR1+ CD8+ T cells population from blood, spleen and lungs at 90 dpi are shown as mean ± SEM, each dot is a mouse. Data are pooled from two experiments and each dot represents one mouse, n≥7. Statistically significant differences are highlighted; *, p < 0.05; **, p < 0.01; ***, p < 0.001; (Mann-Whitney U Test); mean ± SEM values are plotted.</p

Defective NFAT signaling in CD8⁺ T cells leads to accumulation of memory CD8⁺ T cells in LNs.

(A-E) Mixed BMC animals were infected with 106 PFU of MCMV and CD8+ T cell responses were analyzed by tetramer staining and flow cytometry at 7 dpi. (A) Total number of indicated tetramer+ CD8+ T cells in spleen, lungs and mesenteric LN. (B-D) Relative size of CM, effector (B), KLRG1+CD27-, KLRG1+CD27+, KLRG1-CD27+ (C) and CX3CR1+ (D) CD8+ T cell populations from spleen are plotted. (E) Percentages of total CD45.1-/-CD45.2+/+ NFATc1c2 DKO and CD45.1+/-CD45.2+/- WT cells among CD8+ T cells in different organs. (F-I) 104 naïve OTI T cells were transferred to congenic animals and activated by MCMV infection. (F) Absolute count of total OTI T cells in spleen, lungs and different LNs at 7 dpi. (G) WT and NFATc1c2 DKO OTI T cells were isolated from spleen or LNs and transcriptional analysis was performed. Transcriptional profile of genes involved in T cell activation, migration and cell cycle regulation are shown as a heatmap. (H) Flow-cytometric plots showing Ki-67 expression on total CD8+ T cell population (left). Frequency of Ki-67+ cells among total OTI and CM OTI are shown. Panel A-E show data pooled from two experiments (n = 5–6) and panel F has data pooled from 2–3 experiments (n = 5–10). Plots show means ± SEM values, each dot represents one mouse. Statistically significant differences are highlighted; *, p < 0.05; **, p < 0.01; ***, p < 0.001; (Mann-Whitney U Test).</p

Phenotype of inflationary CD8⁺ T cells during chronic infection.

Lymphocytes from spleen (A) or lungs (B) of mice infected for 3 months were stained for CD45.1, CD45.2 and CD8 expression as well as the indicated cell-surface molecules. The plots shown are gated on CD45.1+/-CD45.2+/- CD8+ T cells (shaded histogram) or CD45.1-/-CD45.2+/+ CD8+ T cells (black line) from the same sample. Data are representative of at least six individual mice per stain and two independent experiments. (TIF)</p

NFAT KO mice fail to mount inflationary CD8⁺ T cell response following chronic MCMV infection.

Animals lacking either NFATc1, NFATc2 or both were infected with 106 PFU of MCMV intraperitoneally. (A) Total CD8+ T cells and tetramer specific response kinetics in peripheral blood were tracked. Mean ± SEM are plotted for data pooled from 3 independent experiments (n≥12). Total CD4+ T cells, CD8+ T cells (B) and M38 tetramer+ T cell responses (C) in spleen at 90 dpi. (D) mice were sacrificed at 5 dpi to titrate the virus replication in spleen and lungs. (E) Infected mice were sacrificed 6 months post infection to quantify MCMV latent virus load, which is presented as virus copies per 105 host cells in spleen and salivary glands. Data in panel D and E are pooled from two experiments and each dot represents one mouse. Statistically significant differences are highlighted; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; (Mann-Whitney U Test).</p

NFAT molecules controls CD8⁺ T cell differentiation following acute infection and promote distinct transcriptional signature.

104 naïve OTI T cells were transferred to congenic C57BL/6 mice and activated by acute MCMV infection. CD8+ T cell responses were analyzed at 7 dpi. (A) Absolute number of OTI T cells in blood. (B) Absolute and relative size of CM OTI T cell in blood of acutely infected animals. (C) Frequency of KLRG1+CD27-, KLRG1-CD27+ and KLRG1+CD27+ populations among blood OTI T cells are plotted. (D-H) Transcriptional analysis (RNA sequencing) was performed on OTI T cells isolated from spleen of acutely infected animals (7 dpi). (D) Principal component analysis of all RNA sequencing samples. Replicates of the same group are indicated by the same color as shown in the legend. (E) Volcano plot of genes that are differentially regulated in NFATc1 KO and NFATc2 KO OTI cells. (F) Venn diagram showing the overlap between differentially expressed genes (>2-fold, (G) Top 500 genes differentially regulated between NFATc1c2 DKO and WT OTI T cells were selected and their expression in all groups are shown as heatmap. Transcriptional regulators and genes involved in T cell activation have been annotated. (H) Negative gene-set enrichment of genes associated with effector CD8+ T cells [63] among differentially expressed genes of NFATc1 KO vs WT OTI T cells and NFATc2 KO vs WT OTI T cells. Panel A-C data was pooled from three experiments, where each dot represents one mouse, mean ± SEM values are plotted. Statistically significant differences are highlighted; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; (Mann-Whitney U Test). The statistical comparisons in panel C are between NFAT KO populations and WT counterpart.</p

NFATc1 is crucial for persistent CD8⁺ T cell response during chronic infection.

(A) Overview of mixed bone marrow chimera (BMC) generation, MCMV infection and CD8+ T cell response monitoring. Lethally irradiated C57BL/6J mice were reconstituted with bone marrow mix (1:1) of the control WT BM (CD45.1+/-CD45.2+/-) and either NFATc1 KO, NFATc2 KO, NFATc1c2 DKO or WT BM (CD45.1-/-CD45.2+/+). CD8+ T cell response kinetics were monitored for 90 days following intraperitoneal MCMV infection with 106 PFU. Mice were sacrificed 90 dpi and CD8+ T cell responses in blood and organs were analyzed. (B) Representative flow-cytometric plots showing total CD8+ T cells during acute and chronic infection. (C) Percentage of the CD45.2+/+ subset in the CD8+ T cell population was tracked by labelling peripheral blood. (D) CD8+ T cell response was tracked by labelling peripheral blood with MCMV epitope-specific tetramers. The total number of epitope-specific CD8+ T cells among CD45.2+ population is plotted. (E) Bar plots represent the mean ± SEM of tetramer+ CD8+ T cells for each epitope in spleen, lungs, and mesenteric LN at 90 dpi. Data are pooled from two experiments and each dot represents one mouse, n = 6–10. Statistically significant differences are highlighted; *, p < 0.05; **, p < 0.01; ***, p < 0.001; (Mann-Whitney U Test); mean ± SEM values are plotted.</p

CD8⁺ T cells lacking NFATc1 and NFATc2 showed distinct transcriptional profile.

Naïve OTI T cells (104) were transferred to congenic animals and activated by acute MCMV infection. Animals were sacrificed at 7 dpi and transcriptional analysis was performed. (A) Volcano plot shows genes that are differentially regulated in NFATc1c2 DKO OTI cells as compared to WT cells. (B) Principal component analysis of RNA sequencing samples from WT and NFATc1c2 DKO CD8+ T cells from spleen and lymph nodes (LN). Replicates of the same genotype and tissue are indicated by similar color and shape, respectively. (C) Heatmap shows expression of selected cytokine and cytokine receptor genes in OTI cells that lack NFATc1, NFATc2 or both. (D) Heatmap shows expression of selected transcription factors, surface receptors and cell cycle regulation genes from CD8+ T cells. Color shows Z-score differences. (TIF)</p

Representative gating strategy.

Following singlet gating, lymphocytes were selected with forward/side scatter parameters. Live CD3+ cells were identified by excluding 7AAD stained cells, and CD8+ T cells were selected by CD8a expression. Next, CD8+ T cells (blue gate and arrow) were gated into CD45.2+ single and CD45.1+CD45.2+ double positive populations in mixed bone chimeric animals. Population arising from each bone marrow was progressively gated to define naive and primed CD8+ T cells based on CD44 and CD11a expression. Central memory cells were distinguished from effector cells by CD62L and CD27 expression within primed population. Similarly, short lived effector and memory cell populations (SLEC and Mem) were gated according to KLRG1 and CD27 expression within primed CD8+ T cells (green gate and arrow). Tetramer+ (M38+ and m139+) and CX3CR1+ populations were defined by gating directly on CD45.2+ CD8+ T cells (red gate and arrow) or control CD45.1+CD45.2+ CD8+ T cell populations. (TIF)</p