Human metapneumovirus respiratory infection affects both innate and adaptive intestinal immunity

Abstract

Introduction: Respiratory infections are one of the leading causes of morbidity and mortality worldwide, mainly in children, immunocompromised people, and the elderly. Several respiratory viruses can induce intestinal inflammation and alterations in intestinal microbiota composition. Human metapneumovirus (HMPV) is one of the major respiratory viruses contributing to infant mortality in children under 5 years of age worldwide, and the effect of this infection at the gut level has not been studied.

Methods: Here, we evaluated the distal effects of HMPV infection on intestinal microbiota and inflammation in a murine model, analyzing several post-infection times (days 1, 3, and 5). Six to eight-week-old C57BL/6 mice were infected intranasally with HMPV, and mice inoculated with a non-infectious supernatant (Mock) were used as a control group.

Results: We did not detect HMPV viral load in the intestine, but we observed significant changes in the transcription of IFN-γ in the colon, analyzed by qPCR, at day 1 post-infection as compared to the control group. Furthermore, we analyzed the frequencies of different innate and adaptive immune cells in the colonic lamina propria, using flow cytometry. The frequency of monocyte populations was altered in the colon of HMPV -infected mice at days 1 and 3, with no significant difference from control mice at day 5 post-infection. Moreover, colonic CD8⁺ T cells and memory precursor effector CD8⁺ T cells were significantly increased in HMPV-infected mice at day 5, suggesting that HMPV may also alter intestinal adaptive immunity. Additionally, we did not find alterations in antimicrobial peptide expression, the frequency of colonic IgA⁺ plasma cells, and levels of fecal IgA. Some minor alterations in the fecal microbiota composition of HMPV -infected mice were detected using 16s rRNA sequencing. However, no significant differences were found in β-diversity and relative abundance at the genus level.

Discussion: To our knowledge, this is the first report describing the alterations in intestinal immunity following respiratory infection with HMPV infection. These effects do not seem to be mediated by direct viral infection in the intestinal tract. Our results indicate that HMPV can affect colonic innate and adaptive immunity but does not significantly alter the microbiota composition, and further research is required to understand the mechanisms inducing these distal effects in the intestine.

Keywords: CD8 + T cells; HMPV; lung-gut axis; microbiota; monocytes.

Figure 1

HMPV-infected mice do not exhibit viral load in the intestine at different post-infection times. C57BL/6 mice were infected for 1, 3, and 5 days with HMPV and compared to mice treated with mock. (A) Mouse weight was assessed at different time points of the infection and compared to their initial weight. (B-D) HMPV viral load was assessed using RT-qPCR at different time points post-infection (days 1, 3, and 5) in the lung (B), small intestine (C), and colon (D). Viral load was assessed by comparing to the expression of a host housekeeping gene (β-actin). Mock-treated mice are shown in grey, and HMPV-infected mice are shown in black. All data are shown as mean ± SEM and are representative of two independent experiments (mock-treated mice n=5-6 per group; HMPV-infected mice n=6 per group). Statistical differences were evaluated by a two-way ANOVA comparing the means of all the columns and rows corresponding to each group, followed by a post hoc Tukey test (*p<0.05, ****p<0.0001). ns, not significant.

Figure 2

HMPV-infected mice exhibit increased expression of IFN-γ in the colon at day 1 post-infection. Transcription of different pro-inflammatory cytokines and mucins was assessed in the colon of mock-treated and HMPV-infected mice at different time points. (A) Relative expression of Ifng. (B) Relative expression of Il17a. (C) Relative expression of Il6. (D) Relative expression of Il23a. (E) Relative expression of Muc5ac. (F) Relative expression of Muc2. Mock-treated mice are shown in grey, and HMPV-infected mice are shown in black. All data are shown as mean ± SEM and are representative of two independent experiments (mock-treated mice n=5-6 per group; HMPV-infected mice n=6 per group). Statistical differences were evaluated by a two-way ANOVA comparing the means of all the columns and rows corresponding to each group, followed by a post hoc Tukey test (*p<0.05 and **p<0.01). ns, not significant.

Figure 3

HMPV-infected mice exhibit increased frequency of proinflammatory monocytes in the colon at early infection. Colon lamina propria cells were analyzed at different time points following HMPV infection using flow cytometry. (A) Representative plots of colonic Ly6C⁺ MHCII^-, Ly6C⁺ MHCII⁺, and macrophages (gated as live CD45⁺ CD11b⁺ Ly6G^- SiglecF^- CD64⁺ cells) in mock-treated and HMPV-infected mice at day 5 post-infection. (B) Frequency of Ly6C⁺ MHCII^- monocytes. (C) Frequency of Ly6C⁺ MHCII⁺ monocytes. (D) Frequency of macrophages. Mock-treated mice are shown in grey, and hMPV-infected mice are shown in black. All data are shown as mean ± SEM and are representative of two independent experiments (mock-treated mice n=5-6 per group; HMPV-infected mice n=6 per group). Statistical differences were evaluated by a two-way ANOVA comparing the means of all the columns and rows corresponding to each group, followed by a post hoc Tukey test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). ns, not significant.

Figure 4

HMPV-infected mice exhibit increased frequency of CD8⁺ T cells in the colon at day 5 post-infection. Colon lamina propria T cells were analyzed at different time points following HMPV infection using flow cytometry. (A) Representative plots of CD8⁺ and CD4⁺ T cells (gated as live CD45⁺ CD11b^- CD5⁺ cells) in Mock and HMPV-infected mice at day 5 post-infection. (B) Frequency of CD8⁺ T cells. (C) Frequency of CD4⁺ T cells. (D) Representative plots of memory precursor effector CD8+ T cells (identified as KLRG1^- CD127⁺ cells) and short-lived effector memory CD8+ T cells (identified as KLRG1⁺ CD127^- cells). (E) Frequency of short-lived effector CD8⁺ T cells. (F) Frequency of memory precursor effector CD8⁺ T cells. Mock-treated mice are shown in grey, and HMPV-infected mice are shown in black. All data are shown as mean ± SEM and are representative of two independent experiments (mock-treated mice n=5-6 per group; HMPV-infected mice n=6 per group). Statistical differences were evaluated by a two-way ANOVA comparing the means of all the columns and rows corresponding to each group, followed by a post hoc Tukey test (*p<0.05, **p<0.01, ****p<0.0001). ns, not significant.

Figure 5

Colonic IgA production is not altered following HMPV infection. IgA production was assessed using flow cytometry and ELISA following HMPV infection. (A) Representative plots of colonic IgA⁺ plasma cells (gated as live CD45⁺ MHCII⁺ CD11b^- cells) in mock-treated and HMPV-infected mice at day 5 post-infection. (B) Frequency of colonic IgA⁺ plasma cells. (C) Evaluation of the concentration of free IgA measured by ELISA in feces isolated from HMPV-infected mice at different post-infection times. Mock-treated mice are shown in grey, and HMPV-infected mice are shown in black. All data are shown as mean ± SEM and are representative of two independent experiments (mock-treated mice n=5-6 per group; HMPV-infected mice n=6 per group). Statistical differences were evaluated by a two-way ANOVA comparing the means of all the columns and rows corresponding to each group, followed by a post hoc Tukey test. ns, not significant.

Figure 6

HMPV-infected mice do not exhibit significant alterations in the fecal microbiota. Bacterial DNA from fecal samples was isolated, and 16S rRNA sequencing was performed. Mock and HMPV microbiota were grouped by day post-infection. (A) Microbiota abundance between family members of the phylum Firmicutes. (B) Microbiota abundance between family members of the phylum Bacteroidota. (C) Microbiota abundance at the genus level, showing the 15 most abundant genera at each time point (Kruskal-Wallis, p < 0.05). Mock-treated mice are shown in grey, and HMPV-infected mice are shown in black. All data are shown as mean ± SD and are representative of two independent experiments (mock-treated mice n=5-6 per group; HMPV-infected mice n=6 per group).