Respiratory Tract Epithelial Cells Express Retinaldehyde Dehydrogenase ALDH1A and Enhance IgA Production by Stimulated B Cells in the Presence of Vitamin A

<div><p>Morbidity and mortality due to viral infections are major health concerns, particularly when individuals are vitamin A deficient. Vitamin A deficiency significantly impairs mucosal IgA, a first line of defense against virus at its point of entry. Previous reports have suggested that CD11c^Hi dendritic cells (DCs) of the gastrointestinal tract produce retinaldehyde dehydrogenase (ALDH1A), which metabolizes vitamin A precursors to retinoic acid to support normal mucosal immunity. Given that the upper respiratory tract (URT) and gastrointestinal tract share numerous characteristics, we asked if the CD11c^Hi DCs of the URT might also express ALDH1A. To address this question, we examined both CD11c^Hi test cells and CD11c^Lo/neg control cells from nasal tissue. Surprisingly, the CD11c^Lo/neg cells expressed more ALDH1A mRNA per cell than did the CD11c^Hi cells. Further evaluation of CD11c^Lo/neg populations by PCR and staining of respiratory tract sections revealed that epithelial cells were robust producers of both ALDH1A mRNA and protein. Moreover, CD11c^Lo/neg cells from nasal tissue (and a homogeneous respiratory tract epithelial cell line) enhanced IgA production by lipopolysaccharide (LPS)-stimulated splenocyte cultures in the presence of the retinoic acid precursor retinol. Within co-cultures, there was increased expression of MCP-1, IL-6, and GM-CSF, the latter two of which were necessary for IgA upregulation. All three cytokines/chemokines were expressed by the LPS-stimulated respiratory tract epithelial cell line in the absence of splenocytes. These data demonstrate the autonomous potential of respiratory tract epithelial cells to support vitamin A-mediated IgA production, and encourage the clinical testing of intranasal vitamin A supplements in vitamin A deficient populations to improve mucosal immune responses toward respiratory tract pathogens and vaccines.</p></div

Respiratory tract epithelial cells produce ALDH1A2.

<p>Tissue sections were prepared from the URT and lungs of C57BL/6 mice. Sections were stained with rabbit anti-mouse ALDH1A2 or rabbit anti-goat antibody (control). Bound rabbit anti-mouse ALDH1A2 antibody was detected using Alexa Fluor 568 donkey anti-rabbit IgG. The tissues were counter stained with nuclear stain DAPI. Sections of URT and lung are shown at low (10×) and high (40×) magnification. Staining indicated that the greatest ALDH1A2 protein expression was among epithelial cells lining the respiratory tract.</p

Cytokine production in splenocyte co-cultures.

<p>Spleen cells were stimulated with LPS in the presence of LET cells and/or retinol. Culture components are indicated below each bar. Cytokines that were reproducibly elevated in the presence of LET cells and retinol are shown, including GM-CSF (<b>Panel A</b>), MCP-1 (<b>Panel B</b>), and IL-6 (<b>Panel C</b>). IFNγ levels were reduced in the presence of LET cells and retinol (<b>Panel D</b>). <b>Panel E:</b> IgA levels are shown following splenocyte/LET co-cultures in the presence of LPS and retinol, with and without neutralization using anti-IL-6 and/or anti-GMC-SF antibodies. ‘++’ indicates that antibodies were added to cultures at levels exceeding manufacturers’ recommendations, either 5× (for anti-IL-6 antibodies) or 2× (for anti-GM-CSF antibodies). <b>Panel F:</b> Levels of GM-CSF, MCP-1 and IL-6 are shown in LET cell cultures (without splenocytes) in the presence or absence of LPS.</p

Upregulation of cytokines/chemokines by stimulation of respiratory tract epithelial cells^*.

*<p>LET cells (1×10⁴ cells per well) were cultured with or without LPS. A sample of culture supernatant (25 µl) was tested using bead-based flow cytometry to compare values (pg/ml). Values exceeding 150 pg/ml are bolded.</p

Respiratory tract epithelial cell line enhances IgA production in splenocyte co-cultures.

<p>IgA was tested after culture of splenocytes, LPS, and LET cells in various combinations in the absence (<b>A</b>) or presence (<b>B</b>) of retinol (1 µM). LPS-stimulated splenocytes were also tested in the absence or presence of LET cells, with or without depletion of CD11b⁺/Cd11c⁺ splenocytes (<b>C</b>). <b>Panel D</b> shows IgA expression in cultures of splenocytes or purified T and B cells in the presence of LPS, LET cells and retinol. Culture components are indicated below each bar.</p

URT cells enhance IgA production in splenocyte co-cultures.

<p>Splenocytes were cultured with or without LPS (1 µg/ml) in the absence (<b>A</b>) or presence (<b>B</b>) of retinol (1 µM). Culture components are indicated below each bar. Where indicated, CD11c^Hi and/or CD11c^Lo/neg NT cells were added to cultures. IgA was measured (Y axis, ng/ml) from supernatants after a 7 day culture. Comparisons of IgA in cultures with splenocytes, LPS and retinol with or without CD11c^Lo/neg NT cells revealed a statistically significant difference (p = 0.03). Comparisons of IgA in cultures with splenocytes, CD11c^Lo/neg cells, and LPS with or without retinol also revealed a statistically significant difference (p = 0.04).</p

Working hypothesis for epithelial cell-mediated induction of IgA production in the respiratory tract.

<p>The cartoon illustrates hypothesized mechanisms of vitamin A-dependent IgA production in the respiratory tract. RA = retinoic acid, Macrophage = MΦ.</p

URT cells express ALDH1A mRNA.

<p>RNA was extracted from cells and cDNA was synthesized using oligo-dT₂₀ primers. Serial 1∶10 dilutions of the cDNA were used for PCR amplifications. Gels were loaded from left to right with PCR products from serially diluted cDNA. The left-most columns were representative of products from ∼1×10³ cells. <b>Panel A.</b> Results are shown for NT cells (see Materials and Methods), cervical lymph nodes (CLN) and mesenteric lymph nodes (MesLN) of naïve C57BL/6 mice, separated into CD11c^Hi and CD11c^Lo/neg populations and tested for ALDH1A2 and GAPDH mRNA. <b>Panel B.</b> CD11c^Hi and CD11c^Lo/neg NT populations were tested for ALDH1A1, ALDH1A2, ALDH1A3 and GAPDH mRNA. <b>Panel C.</b> Cells were tested for ALDH1A2 mRNA. Samples included NT cells that had been FACS-sorted for the F4/80⁺CD11c^-CD11b⁺ phenotype (abbreviated ‘F4/80+CD11c-’), two macrophage lines MAC INF4.29 and LIE 13–14, NT cells or lung cells enriched for epithelium by negative selection and short-term culture (see Materials and Methods), and LET cells. On a per-cell basis, the highest ALDH1A expression levels were among CD11c^Lo/neg cell populations.</p

Respiratory Syncytial Virus Human Experimental Infection Model: Provenance, Production, and Sequence of Low-Passaged Memphis-37 Challenge Virus

<div><p>Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in children and is responsible for as many as 199,000 childhood deaths annually worldwide. To support the development of viral therapeutics and vaccines for RSV, a human adult experimental infection model has been established. In this report, we describe the provenance and sequence of RSV Memphis-37, the low-passage clinical isolate used for the model's reproducible, safe, experimental infections of healthy, adult volunteers. The predicted amino acid sequences for major proteins of Memphis-37 are compared to nine other RSV A and B amino acid sequences to examine sites of vaccine, therapeutic, and pathophysiologic interest. Human T- cell epitope sequences previously defined by <i>in vitro</i> studies were observed to be closely matched between Memphis-37 and the laboratory strain RSV A2. Memphis-37 sequences provide baseline data with which to assess: (i) virus heterogeneity that may be evident following virus infection/transmission, (ii) the efficacy of candidate RSV vaccines and therapeutics in the experimental infection model, and (iii) the potential emergence of escape mutants as a consequence of experimental drug treatments. Memphis-37 is a valuable tool for pre-clinical research, and to expedite the clinical development of vaccines, therapeutic immunomodulatory agents, and other antiviral drug strategies for the protection of vulnerable populations against RSV disease.</p></div

Comparison of human CD4 and CD8 T-lymphocyte epitope sequences between respiratory syncytial virus Memphis-37 and A2 strains.

<p>Legend: *Differences between Memphis-37 and RSV A2 are bolded.</p><p>Comparison of human CD4 and CD8 T-lymphocyte epitope sequences between respiratory syncytial virus Memphis-37 and A2 strains.</p