Natural Microbial Exposure from the Earliest Natural Time Point Enhances Immune Development by Expanding Immune Cell Progenitors and Mature Immune Cells

Abstract

Microbial experience fundamentally shapes immunity, particularly during the perinatal period when the immune system is underdeveloped, and novel microbial encounters are common. Most animal models are raised in specific pathogen-free (SPF) conditions with relatively uniform microbial communities. How SPF housing conditions alter early-life immune development relative to natural microbial exposure (NME) has not been thoroughly investigated. In this article, we compare immune development in SPF-raised mice with mice born from immunologically experienced mothers in microbially diverse environments. NME induced broad immune cell expansion, including naive cells, suggesting mechanisms besides activation-induced proliferation contribute to the increase in immune cell numbers. We found NME conditions also expanded immune cell progenitor cell populations in the bone marrow, suggesting microbial experience enhances immune development at the earliest stages of immune cell differentiation. Multiple immune functions characteristically impaired in infants were also enhanced by NME, including T cell memory and Th1 polarization, B cell class switching and Ab production, proinflammatory cytokine expression, and bacterial clearance after Listeria monocytogenes challenge. Collectively, our studies reveal numerous impairments in immune development in SPF conditions relative to natural immune development.

Figure 1:. NME Breeding.

(A) Adult female C57BL/6 mice (green) are cohoused with pet store mice (white) for >3 weeks prior to breeding. Dam and litter remained with pet store mice for up to 6 wks after parturition. (B) Litter sizes (from 61 SPF litters and 14 NME litters) and (C) weight at weaning were measured (collected from 51 SPF litters and 10 NME litters). Data was collected over the course of one year of breeding. **p ≤ 0.01, ****p ≤ 0.0001.

Figure 2:. NME induces broad immune cell expansion in the lymph nodes.

Lymph nodes were harvested from SPF (black) and NME (green) offspring at 2, 3, or 6 weeks of age and split in half for staining with two flow panels. (A) Leukocytes were identified by CD45.2 expression and enumerated. Number and frequency of eight immune cell types as indicated: (B) Ɣδ T cells, (C) B cells, (D) neutrophils, (E) monocytes/macrophages, (F) DCs, (G) NK cells, (H) CD4 T cells, and (I) CD8 T cells. (J) Leukocyte composition. 10-15 mice/group/time point. Data were collected from three individual experiments per group per age over two years. Example flow plots are from a 6-week-old NME mouse. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Figure 3:. NME increases T cell memory commitment.

Number and frequency of (A) naïve, (B) T_cm, and (C) T_em/T_eff CD8 T cells in the lymph nodes harvested from SPF (black) and NME (green) offspring at 2, 3, or 6 weeks of age. (D) Lymph node CD8 T cell composition. Number and frequency of (E) naïve, (F) T_cm, and (G) T_em/T_eff CD4 T cells in the lymph node. (H) Lymph node CD4 T cell composition. 10-15 mice/group/time point. Data were collected from three individual experiments per group per age over two years. Example flow plots are from a 6-week-old NME mouse. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Figure 4:. NME enhances helper T cell polarization.

Number and frequency of (A) T_reg, (B) T_h1, (C) T_h17, (D) T_h2, (E) T_fh CD4 T cells in the lymph node harvested from SPF (black) and NME (green) offspring at 2, 3, or 6 weeks of age. (F) Lymph node CD4 T cell lineage composition. (G) Lymph node T_h1 and T_h17 to T_reg, T_h2, and T_fh ratio. 10-15 mice/group/time point. Data were collected from three individual experiments per group per age over two years. Example flow plots are from a 6-week-old NME mouse. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Figure 5:. Monocyte/macrophage subsets.

Number and frequency of (A) Ly6C⁺ MHCII⁻ (P1), (B) Ly6C⁺ MHCII^int (P2), (C) Ly6C⁻ MHCII⁻ (P4), (D) Ly6C^lo MHC^int (P3a), (E) Ly6C^lo MHCII^hi (P3b) in the lymph node harvested from SPF (black) and NME (green) offspring at 2, 3, or 6 weeks of age. (F) Lymph node monocyte/macrophage composition. 10-15 mice/group/time point. Data were collected from multiple experiments over two years. Example flow plots are from a 6-week-old NME mouse. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Figure 6:. NME increases B cell activation.

Number and frequency of (A) mature activated, (B) naïve, (C) class-switched, (D) and immature B cells in the lymph node harvested from SPF (black) and NME (green) offspring at 2, 3, or 6 weeks of age. (E) Lymph node B cell composition. (F) Number and frequency of plasma cells in the lymph node. 10-15 mice/group/time point. Data were collected from three individual experiments per group per age over two years. Example flow plots are from a 6-week-old NME mouse. (G) Total IgG concentration (ng/mL) in the plasma of 3-week-old SPF and NME mice measured by a single ELISA, samples were assayed in duplicate. 3-4 mice/group. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Figure 7.. NME enhances hematopoiesis.

(A) Hierarchy of differentiation for immune cell progenitors. Bone marrow was harvested from SPF (black) and NME (green) pups at day of life (DOL) 3 and (B) LSK (Lin⁻ Sca-1⁺ c-Kit⁺), (C) HSC (Lin⁻ Sca-1⁺ c-Kit⁺ Flt-3⁻ CD34⁻), (D) MPP4/LMPP (Lin⁻ Sca-1⁺ c-Kit⁺ Flt-3^hi), (E) GMP (Lin⁻ Sca-1⁻ c-Kit⁺ CD16+ CD34+), (F) MEP (Lin⁻ Sca-1⁻ c-Kit⁺ CD16⁻ CD34⁻), (F) CLP (Lin⁻ Sca-1⁻ c-Kit⁻ Flt3⁺) were enumerated flow cytometrically. 8-14 mice/group. Data was collected from 2-3 separate experiments. Example flow plots are from a DOL 3 NME mouse. Statistical analyses were determined using Mann-Whitney Test.

Figure 8:. NME enhances cytokine, PRR expression, and host defense.

(A) Cytokine and chemokine levels in plasma harvested from 3-week-old SPF (solid black line), 3-week-old NME (solid green line), 6-week-old SPF (dotted black line), and 6-week-old NME (dotted green line) mice. Data depicts the average of 13 mice and two experimental replicates. (B) qPCR analysis of TLRs, MyD88, and CD14 in adherent myeloid cells isolated from spleens of SPF (n = 4) and NME (n = 8) mice. Data are representative of 2 technical replicates. (C) LM CFU in spleen 4 days post inoculation in SPF (black) and NME (green) mice. 9 mice/group were analyzed, representative of 2 technical replicates. ^†p < 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.