Presentation_1_Myeloid- and epithelial-derived RELMα contribute to tissue repair following lung helminth infection.pptx

Soil-transmitted helminth (STH) infections impact billions of individuals globally; however, there is a need to clarify the long-term impacts of these infections on pulmonary health owing to their transient migration and subsequent damage to the lungs. In mouse models of these infections using Nippostrongylus brasiliensis, lung pathology persists at later time points post single infection. These studies also indicate the persistent transcriptional expression of resistin-like molecule α (RELMα), an immunomodulatory protein induced in type 2 immunity and alternatively activated macrophages. Using constitutive and tamoxifen-inducible cell-specific RELMα knockout mouse strains, we identified that epithelial- and myeloid-derived RELMα protein remained elevated at 30 days post infection and altered the immune cell signature and gene expression in lung compartments. Histopathological assessment of alveolar damage revealed a role for RELMα in tissue repair, suggesting the importance of sustained RELMα expression for lung recovery from helminth infection. Acellular three-dimensional (3D) lung scaffolds were prepared from the lungs of wild-type (WT), RELMα KO-naive, or 30 days post N. brasiliensis-infected mice to assess their ability to support epithelial cell growth. N. brasiliensis infection significantly altered the scaffold and impaired epithelial cell growth and metabolic activity, especially in the RELMα KO scaffolds. These findings underscore a need to identify the long-term impacts of helminth infection on human pulmonary disease, particularly as alveolar destruction can develop into chronic obstructive pulmonary disease (COPD), which remains among the top global causes of death. Translation of these findings to human protein resistin, with sequence homology to RELMα therapeutic opportunities in lung repair.</p

Intestinal measurements of WT mice following infection with WT- or STAT6 KO-adapted <i>N</i>. <i>brasiliensis</i>.

A. Length of intestine. B-E. RT-qPCR expression relative to GAPDH. Circles are individual infected hosts, bars are mean values, ns p-value > 0.05 by t-test. Data is representative of 1 independent experiment. (TIF)</p

Complete GSEA results in G1 day 5 adult worms from WT or STAT6 KO mice.

Complete GSEA results in G1 day 5 adult worms from WT or STAT6 KO mice.</p

Comparison of differentially expressed genes in adapted (G15/14) vs G1 adult female <i>N</i>. <i>brasiliensis</i> from STAT6 vs WT hosts.

A. Venn diagrams of significantly up (Log2FoldChange > 2) versus down (Log2FoldChange B. Scatterplot of all STAT6 KO vs. WT host condition Log2(FoldChange) values, filtered on adjusted p-value N. brasiliensis infection, (G1) (y-axis) versus adapted N. brasiliensis (G15/14) (x-axis). Values represent the number of genes in each quadrant. (TIF)</p

<i>N</i>. <i>brasiliensis</i> exposed to STAT6 KO hosts for one prior generation do not elicit a stronger inflammatory WT host immune response.

Mesenteric lymph node data (A-J) A. Experimental design; Parental worms infected WT or STAT6 KO mice, and resulting progeny (G1 adapted) infected WT mice. Flow cytometry was run on cells from mesenteric lymph nodes at day 8 post infection. B. Total number of mesenteric lymph node cells. C-D. Percent and total cell number of IL-13+ Th2 cells. E-F. Percent and total cell number of ST2+ Th2 cells. G-H. Percent and total cell number of IL-17+ γδT cells. I-J. Percent and total cell number of IFNγ+ CD8+ T cells in mesenteric lymph nodes. Data from the peritoneal cavity (K-P). K-L. Percentage of parent population or total cell number of macrophages. M-N. Percentage or total cell number of Arg-1 expressing macrophages. O-P. Percentage of parent population of eosinophils and neutrophils. Q. Parasite burden by number of eggs per gram of feces (EPG) for each day post infection. R. Area under the curve. S. Number of worms from host small intestine at day 8 post infection. Each point is a replicate mouse, and error bars are SEM. * p (TIF)</p

Parasitology associated with WT mice infected with WT or STAT6 KO adapted <i>N</i>. <i>brasiliensis</i> (G25/24) Fig 6.

A. Average eggs per gram of feces by days post infection, with SEM error bars. B. Average area under the curve of data plotted in A. C. Number of adult parasites in the small intestine at day 8 post infection. (TIF)</p

Fig 6 -

STAT6 KO-adapted N. brasiliensis elicit a stronger inflammatory WT host immune response. Mesenteric lymph node data (A-K) A. Experimental design; WT or STAT6 KO (generation 25 or 24, respectively) adapted worms infected WT mice, and flow cytometry was run on cells from mesenteric lymph nodes at day 8 post infection. B. Gating strategy. C. Total number of mesenteric lymph node cells. D-E. Percent and total cell number of IL-13+ Th2 cells. F-G. Percent and total cell number of ST2+ Th2 cells. H-I. Percent and total cell number of IL-17+ γδT cells. J-K. Percent and total cell number of IFNγ+ CD8+ T cells in mesenteric lymph nodes. Data from the peritoneal cavity (L-T). L. Gating strategy to detect myeloid cell populations; cell types outlined by boxes. M-N. Percentage of parent population or total cell number of FcεR positive cells. O-P. Percentage of parent population or total cell number of macrophages. Q-R. Percentage or total cell number of Arg-1 expressing macrophages. S-T. Percentage of parent population of eosinophils or neutrophils. Each point is a replicate mouse, and error bars are SEM. * p < 0.05 ** p < 0.01, *** p < 0.001, by t-test. Data are representative of 3 independent experiments.</p

WT host survival decreased with STAT6 KO-adapted <i>N</i>. <i>brasiliensis</i> infection.

A. Survival curve with average host survival by days post infection for WT hosts infected with WT-adapted or STAT6 KO-adapted worms. Combined data from 9 independent experiments, 49 mice per condition, Mantel-Cox test p-value. (TIF)</p

Extended list of key reagents.

The impact of the host immune environment on parasite transcription and fitness is currently unknown. It is widely held that hookworm infections have an immunomodulatory impact on the host, but whether the converse is true remains unclear. Immunity against adult-stage hookworms is largely mediated by Type 2 immune responses driven by the transcription factor Signal Transducer and Activator of Transcription 6 (STAT6). This study investigated whether serial passage of the rodent hookworm Nippostrongylus brasiliensis in STAT6-deficient mice (STAT6 KO) caused changes in parasites over time. After adaptation to STAT6 KO hosts, N. brasiliensis increased their reproductive output, feeding capacity, energy content, and body size. Using an improved N. brasiliensis genome, we found that these physiological changes corresponded with a dramatic shift in the transcriptional landscape, including increased expression of gene pathways associated with egg production, but a decrease in genes encoding neuropeptides, proteases, SCP/TAPS proteins, and transthyretin-like proteins; the latter three categories have been repeatedly observed in hookworm excreted/secreted proteins (ESPs) implicated in immunosuppression. Although transcriptional changes started to appear in the first generation of passage in STAT6 KO hosts for both immature and mature adult stages, downregulation of the genes putatively involved in immunosuppression was only observed after multiple generations in this immunodeficient environment. When STAT6 KO-adapted N. brasiliensis were reintroduced to a naive WT host after up to 26 generations, this progressive change in host-adaptation corresponded to increased production of inflammatory cytokines by the WT host. Surprisingly, however, this single exposure of STAT6 KO-adapted N. brasiliensis to WT hosts resulted in worms that were morphologically and transcriptionally indistinguishable from WT-adapted parasites. This work uncovers remarkable plasticity in the ability of hookworms to adapt to their hosts, which may present a general feature of parasitic nematodes.</div

Heatmaps of gene expression data from WT or STAT6 KO (G 9/7) adapted female worms derived from the intestine at day 7 post infection of WT mice.

A. Heatmap and GSEA enrichment plot for genes encoding adult stage ESP-associated gene categories. B. Heatmaps for expression of genes in Chromosome, Gene Silencing, or FMRFamide-like families. (TIF)</p