Equine keratinocytes in the pathogenesis of insect bite hypersensitivity: Just another brick in the wall?

Equine insect bite hypersensitivity (IBH) is the most common skin disease affecting horses. It is described as an IgE-mediated, Type I hypersensitivity reaction to salivary gland proteins of Culicoides insects. Together with Th2 cells, epithelial barrier cells play an important role in development of Type I hypersensitivities. In order to elucidate the role of equine keratinocytes in development of IBH, we stimulated keratinocytes derived from IBH-affected (IBH-KER) (n = 9) and healthy horses (H-KER) (n = 9) with Culicoides recombinant allergens and extract, allergic cytokine milieu (ACM) and a Toll like receptor ligand 1/2 (TLR-1/2-L) and investigated their transcriptomes. Stimulation of keratinocytes with Culicoides allergens did not induce transcriptional changes. However, when stimulated with allergic cytokine milieu, their gene expression significantly changed. We found upregulation of genes encoding for CCL5, -11, -20, -27 and interleukins such as IL31. We also found a strong downregulation of genes such as SCEL and KRT16 involved in the formation of epithelial barrier. Following stimulation with TLR-1/2-L, keratinocytes significantly upregulated expression of genes affecting Toll like receptor and NOD-receptor signaling pathway as well as NF-kappa B signaling pathway, among others. The transcriptomes of IBH-KER and H-KER were very similar: without stimulations they only differed in one gene (CTSL); following stimulation with allergic cytokine milieu we found only 23 differentially expressed genes (e.g. CXCL10 and 11) and following stimulation with TLR-1/2-L they only differed by expression of seven genes. Our data suggests that keratinocytes contribute to the innate immune response and are able to elicit responses to different stimuli, possibly playing a role in the pathogenesis of IBH.

Introduction

Equine insect bite hypersensitivity is the most common skin disease affecting horses [1]. IBH is caused by Type I hypersensitivity to the bites of Culicoides midges and resembles human atopic dermatitis (AD) [2-4]. The most pronounced clinical sign of IBH is pruritus [5]. Affected horses develop skin lesions that are most commonly distributed along the dorsal midline, in particular under the mane and around the tail, and less often on the ventral midline, on the head and legs, depicting preferred feeding sites of Culicoides insects [4,6]. Skin lesions are initially characterized as papules and edema, which due to strong pruritus and inflicted self-trauma further develop into alopecia and excoriation, followed by acanthosis and lichenification [7].

Immunologically, equine IBH is described as an IgE mediated, Type I hypersensitivity reaction to salivary gland proteins of Culicoides insects [8-10]. While feeding, Culicoides cause significant mechanical damage to the skin and inject a pool of various salivary gland proteins which act as allergens in predisposed horses [11]. Many of these allergens are enzymes such as proteases, hyaluronidase and maltase, while the biological function of others is still not known. Allergens from three different Culicoides species, C. nubeculosus, C. sonorensis and C. obsoletus have been identified and produced as recombinant proteins [12-16]. All of the allergens have been expressed in E. coli, some in insect cells, barley and in P. pastoris [16-18]. Although production of recombinant proteins in E. coli is the most common, it bears many disadvantages for use in cellular assays, such as endotoxin contamination and lack of post-translational modification of the protein, leading to unspecific stimulation or lack of response, respectively, and thus limiting their use for cellular in vitro assays [18].

Type I hypersensitivities develop as a result of activation of T helper type 2 cells (Th2) and their signature cytokines IL-4, IL-5 and IL-13 [19]. These cytokines are responsible for production of allergen specific IgE antibodies by B cells that bind to the IgE high affinity receptor, FcεRI, expressed on mast cells. The binding of IgE to the FcεRI and crosslinking of bound IgE with allergen activates mast cells and causes the release of pro-inflammatory mediators [19-21]. Additionally, a line of recent evidence has shown that epithelial barriers play a major role in development of Type I hypersensitivities alongside Th2 cells [22-24]. In humans suffering from AD, null variants in filaggrin, a protein involved in terminal differentiation of keratinocytes, severely disrupts the epithelial barrier, thus predisposing individuals with such mutation to AD [25]. Moreover, keratinocytes have been shown to have a high immunological potential as they can produce cytokines such as thymic stromal lymphopoietin (TSLP), IL-33 and IL-25 [24,26]. These cytokines have been demonstrated to play a major role in early development of allergic response [27,28]. Nevertheless, the initiating factors that lead to a Th2 immune response are not completely elucidated yet. The expression of TSLP in human keratinocytes is induced by different Toll like receptor (TLR) ligands, as well as by allergic cytokine milieu [29-31]. Accordingly, activation of keratinocytes can also occur as a consequence of a local Th2 environment. Allergic cytokine milieu (ACM), produced by Th2 lymphocytes and allergic inflammatory cells, consists of IL-4, IL-5, IL-13 and TNF-α [27]. This ACM is found in human allergic individuals upon activation of Th2 immune response. In horses, injection of C. obsoletus allergens in the skin induced a local increase of IL-4, confirming the importance of this cytokine in IBH [32].

Recently, we have also shown that equine keratinocytes respond to different TLR ligands, in particular TLR 1/2 ligand, by upregulation of TSLP mRNA. Furthermore, TLSP was upregulated after stimulation with an ACM consisting of a combination of recombinant equine IL4 and TNF-α [33].

Although associations between epithelial barrier disruption and development of Type I hypersensitivities have been thoroughly studied in human patients, the exact role of keratinocytes in the pathogenesis of Type I hypersensitivities is not entirely understood. Based on similarities in the pathogenesis of IBH and AD, this equine skin disease represents a valuable source of information from horses with spontaneously occurring disease for the role of keratinocytes in allergic skin diseases also for other species. We have recently reported that lesional skin of IBH horses is transcriptionally characterized by disruption of the epithelial barrier and a strong immune cell transcriptional signature [34]. Moreover, we have demonstrated that the non-lesional epidermis of IBH-affected horses differs transcriptionally from the epidermis of healthy horses by changes in lipid metabolism and a propensity to develop itch, which is the cardinal clinical sign of IBH, suggesting an involvement of the epithelial barrier in development of IBH [34].

Therefore, we aimed at understanding how keratinocytes react to stimulation with Culicoides allergens. Moreover, we aimed to investigate how keratinocytes respond to ACM alone or in addition to these allergens. We also studied their response to stimulation with a Toll like receptor 1/2 synthetic ligand, Pam3CSK4, as IBH lesions are sometimes further exacerbated by secondary bacterial infections. Lastly, we wanted to investigate whether the responses differ between keratinocytes derived from IBH-affected or from healthy horses.

Materials and methods

Sample collection

This study was approved by the Animal experimental Committee of the Canton of Bern, Switzerland (No. BE 69/18). IBH-affected horses were diagnosed based on recurrent clinical signs of IBH. Diagnosis of IBH was additionally confirmed by histological examination [34]. Samples were collected from 8 horses slaughtered due to IBH and one clinical patient suffering from IBH. In the clinical patient, two 8mm punch biopsies were taken from non-lesional skin of the inner thigh after sedation with detomidine hydrochloride (0.01 mg/kg iv; Domosedan, www.vetoquinol.ch) and local subcutaneous injection of lidocaine. 5 x 5 cm skin pieces were taken from the inner thigh of IBH-affected slaughtered horses. Skin samples were collected from the same region in 9 slaughtered control horses with no apparent skin diseases and no clinical history of skin diseases. All samples were taken immediately after slaughter. All skin samples and biopsies were transported in pre-cooled Williams E medium on ice to the laboratory where they were processed immediately (S1 Table). Written informed owner consent was obtained from the owner of the patient.

Isolation and culture of primary equine keratinocytes

Isolation and culture of keratinocytes was performed using a dispase II-based skin digestion protocol as in Cvitas et al [33]. Briefly: skin samples were incubated at 4°C for 24h with 10 mg/ml Dispase II (Roche, Basel, Switzerland) in Williams E medium (Bioconcept, Allschwil, Switzerland). Subsequently, the epidermis was separated from the dermis and further digested in accutase (CELLnTEC, Bern, Switzerland) for 20 min at room temperature. Only keratinocytes derived from non-lesional skin were obtained, as detachment of the epidermis without fibroblast contamination did not work out with lesional skin in our hand. Keratinocytes were seeded at 12 x 103 cells per cm2 of cell culture flask and grown in complete Williams E medium. Cells were cultured in 75 cm2 flasks at density of 9 x 105 cells per flask; at 35°C, 5% CO2 until they reached 90% confluence and were then passed. Cells of passage three were used for stimulation experiments. After reaching 80% confluence, the cells were incubated with different stimulation conditions for 24h at 35°C, 5% CO2 (Tables 1 and S1).

Table 1

Stimuli used in the study.

Stimuli	Concentration	Expression system
Pam3CSK41	5 μg/ml	N.A.
Recombinant Culicoides allergen pool:	0.5 μg/ml of each
Cul o 22 (Hyaluronidase)	0.5 μg/ml	P. pastoris
Cul o 32 (PR-1 like; Antigen-5 like)	0.5 μg/ml	P. pastoris
Cul n 42 (Unknown)	0.5 μg/ml	P. pastoris
Cul o 72 (Unknown)	0.5 μg/ml	P. pastoris
Cul n 82 (Maltase, Alpha amylase)	0.5 μg/ml	P. pastoris
C. nubeculosus whole body extract	5 μg/ml	N.A.
Recombinant equine IL-43	100 ng/ml	P. pastoris
Recombinant equine TNF-α4	100 ng/ml	E. coli

1 Invivogen, San Diego, California, USA.

2 Kindly provided by Boehringer Ingelheim, Ingelheim am Rhein, Germany.

3 KingFisher Biotech, Inc., St. Paul, Minnesota, USA.

4 R&D Systems, Inc., Minneapolis, Minnesota, USA.

Immunofluorescence

At the third passage, keratinocytes were seeded in chambered cell culture slides (Sarstedt, Nümbrecht, Germany) and cultured until they reached 80% confluence. Subsequently, immunofluorescence staining using polyclonal rabbit anti-bovine cytokeratin (Agilent, Santa Clara, California, USA) and mouse monoclonal anti-human vimentin (Agilent) was carried out as described previously [33]. To confirm that our keratinocyte cultures were fibroblast free, the staining was carried out for all cultures used in this study as described [33] (Fig 1).

Fig 1

Primary equine keratinocyte culture: Primary equine keratinocytes were stained with anti-cytokeratin.

(A) and (C) Staining of primary keratinocyte cultures with anti-cytokeratin: Cytoplasmic cytokeratin is shown in pink; nuclei were counterstained with Hoechst and are shown in blue; (A) 20x magnification; (C) 40x magnification. (B) and (D) Staining of keratinocytes with anti-vimentin: No staining was observed with this antibody. Nuclei are shown in blue; (B) 20x magnification; (D) 40x magnification.

Culicoides nubeculosus whole body extract and recombinant allergens

Culicoides nubeculosus (C. nubeculosus) whole body extract (WBE) was prepared as described previously and was sterile filtrated before being used in the keratinocyte cultures [35]. Five recombinant (r-) Culicoides allergens (Table 1) kindly provided by Boehringer-Ingelheim, had been expressed in yeast (Pichia pastoris, Validogen GMBH, (formerly VTU Technology), Grambach, AT) and purified [36].

Stimulation of keratinocytes

Primary keratinocytes derived from IBH-affected horses (IBH-KER) and healthy control horses (H-KER) of passage three were stimulated once they reached 80% confluence. IBH-KER and H-KER were cultured in medium only or with a pool of r-Culicoides allergens, C. nubeculosus WBE or toll like receptor 1/2 synthetic ligand, Pam3CSK4. Additionally, a combination of recombinant equine TNF-α (R&D Systems, Minneapolis, Minnesota, USA) and recombinant equine IL-4 (LubioScience, Zürich, Switzerland) was added to primary equine keratinocytes cultured in the presence or absence of the pool of r-Culicoides allergens and C. nubeculosus WBE (Table 1). The concentration of the ligands used in the study was based on previous work [34,37], while concentrations of the recombinant Culicoides allergens used were based on previously published studies [11,38].

An experimental overview of the stimulations is given in Fig 2 and the details of different stimulation conditions in S1 Table.

Fig 2

Summary of the experimental setup.

Isolation of RNA

Total RNA was isolated from the cultured keratinocytes using RNeasy Mini Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. Prior to RNA extraction, cell lysates were loaded onto a spin column (QIAshredder, Qiagen) and centrifuged at 16’000x g for 2 minutes (Qiagen). Contaminating genomic DNA was removed by on-column DNase treatment, and RNA was quantified spectrophotometrically at 260 nm (NanoDrop 2000c, ThermoScientific, Reinach, Switzerland). Samples were subsequently handed to the Next Genome Sequencing platform of the University of Bern for RNA sequencing. RNA quality was determined using Fragment Bioanalyzer (Labgene, Châtel-Saint-Denis, Switzerland).

RNA sequencing

Illumina TruSeq stranded mRNA libraries were prepared according to the manufacturer’s protocol (Illumina, San Diego, USA). Between 17–31 mil 2 x 50 bp read-pairs per sample were generated on an Illumina NovaSeq 6000 instrument. The quality of the RNA-seq data was assessed using fastqc v. 0.11.5 and RSeQC v. 2.6.4.

Mapping to reference genome and differential gene expression analysis

Differential gene expression analysis was performed as described in Cvitas et al. [34]. Briefly, reads were mapped to the reference genome (EquCab3.0) using HiSat2 v. 2.1.0 and FeatureCounts v. 1.6.0 was used to count the number of reads overlapping with each gene as specified in the genome annotation (NCBI Equus caballus Annotation Release 103). The Bioconductor package DESeq2 v. 1.18.1 was used for differential gene expression analysis.

To test for differential gene expression between the experimental groups we combined the factors “group” (IBH-affected or control) and “treatment” (unstimulated or six different keratinocyte stimulations) into a single factor with all combinations of the original factors (e.g. ibh_unstimulated for samples from unstimulated cells of IBH horses, h_unstimulated for a sample from unstimulated cells of healthy horses etc.) as described in the DESeq2-Vignette (http://bioconductor.org/packages/devel/bioc/vignettes/DESeq2/inst/doc/DESeq2.html#interactions). This resulted in a factor with 14 different combinations/levels (2 groups x 7 treatments) which we used to specify the comparisons i.e., contrast.

The Benjamini Hochberg method was used to correct for multiple testing. We did not remove any genes with low or no expression before running the DESeq analysis as the tool’s “result” function performs an “independent filtering” by default which is based on the mean of normalized counts (see DESeq2 documentation on Bioconductor). Genes with a false discovery rate (= p adjusted) smaller than 0.05, and log2 fold change >1 were considered significantly differentially expressed. The datasets generated during the current study are available in the ENA repository via accession numbers PRJEB37568.

Gene ontology analysis

TopGo v. 2.24.0 was used to identify gene ontology terms significantly enriched for differentially expressed genes (threshold for genes to be significantly differentially expressed: padjusted < 0.05). All tests were repeated using different combinations of algorithm (weight01 or classic) and test statistic (Fisher or Kolmogorov-Smirnov) to assess the robustness of the results. An interactive Shiny application was set up to facilitate the exploration and visualisation of the RNA-seq analysis results. All analyses were run in R version 3.4.4 (2018-03-15).

Pathway analysis

ClusterProfiler v3.10.1 was used to test for enrichment of KEGG pathways with significantly differentially expressed genes. Gene set enrichment analysis (GSEA) was performed using the gseKEGG-function (default settings except for minGSSize = 50) and a ranked list as input (entrezgene-id and it’s corresponding–log (raw pvalue), list sorted according to–log (raw pvalue).

Results

Sequencing data was generated from all of the samples, with exception of samples of three IBH-KER and four H-KER cultures stimulated with Pam3CSK4, as these libraries did not produce enough sequencing reads. Therefore, we only performed the transcriptome analysis with six IBH-KER and five H-KER Pam3CSK4-stimulated cultures. Data derived from one control horse was excluded from the analyses because it had a different expression profile than other horses, and we found expression of some genes that cells of the epithelial origin should not express, suggesting possible contamination of the sample.

Stimulation of primary keratinocytes with Culicoides allergens did not induce changes in their gene expression

In order to investigate whether and how primary keratinocytes possibly contribute to pathogenesis of equine IBH, we stimulated keratinocytes with C. nubeculosus WBE and the pool of r-Culicoides allergens.

When comparing transcriptomes of IBH-KER and H-KER stimulated with WBE or r-Culicoides allergens to unstimulated IBH-KER and H-KER, we found no differentially expressed genes (DEGs; Fig 3A–3D). This was already noticeable in the results of the principal component analysis (PCA) based on 500 most variable genes, where samples of keratinocytes stimulated with Culicoides allergens clustered closely with non-stimulated keratinocytes (S1 Fig).

Fig 3

A-F. Volcano plots of significant DEGs in following comparisons: Non-stimulated (NS) vs. recombinant allergen pool stimulated, in IBH-KER (A) and H-KER (B). NS vs. C. nubeculosus WBE stimulated in IBH-KER (C) and H-KER (D). NS vs. ACM stimulated in IBH-KER (E) and H-KER (F).

Stimulation of primary keratinocytes with the allergic cytokine milieu is characterized by transcriptional changes in immune signatures and epithelial barrier

In order to understand how an allergic microenvironment might affect keratinocytes, we stimulated IBH-KER and H-KER with a combination of recombinant equine IL-4 and TNF-α, mimicking an allergic inflammatory milieu. When comparing IBH-KER stimulated with ACM to non-stimulated IBH-KER, we found 657 DEGs. Three hundred and seventeen (317) DEGs were significantly upregulated and 340 were significantly downregulated (Fig 3E). In H-KER, 413 significantly upregulated and 299 significantly downregulated DEGs were found (Fig 3F). Hierarchical clustering of non-stimulated samples and stimulated samples based on top 30 DEGs showed a clearly separated clustering of samples based on the culture conditions, in both IBH-KER and H-KER. 80% of the top 30 DEGs were shared between IBH-KER and H-KER. Genes involved in the inflammatory response (NFKB1, ROR1, CXCL8), cytokine mediated signaling (IL31, IL23A, CISH) as well as epithelial barrier formation (KRT80, KRT7) were among the top 30 DEGs (Fig 4A and 4B).

Fig 4

A-B Hierarchical clustering gene expression of top 30 genes of allergic cytokine milieu-stimulated and non-stimulated samples from the two compared conditions in both IBH-KER (A) and H-KER (B). Lower mean counts are shown in light green and higher mean counts in dark blue.

Gene ontology (GO) analysis of DEGs between non-stimulated and ACM stimulated IBH-KER indicated enrichment of processes such as inflammatory response, cytokine- and chemokine-mediated signaling and, interestingly, processes of keratinocyte differentiation, hair follicle development and regulation of hair follicle development (Tables 2 and S2).

Table 2

Selected biological processes enriched in IBH-KER stimulated with allergic cytokine milieu in comparison to non-stimulated IBH-KER.

GO-ID	Term	Annotated	Significant	Expected	Classic Fisher
GO:0007229	Integrin-mediated signaling	71	34	18.46	5.80E-05
GO:0030593	Neutrophil chemotaxis	46	21	11.96	0.00042
GO:0070098	Chemokine-mediated signaling	34	17	8.84	0.0012
GO:0019885	Antigen processing and presentation	8	7	2.08	0.00043
GO:0006954	Inflammatory response	298	109	77.48	3.00E-05
GO:0019221	Cytokine-mediated signaling	252	109	65.52	1.50E-09
GO:0010634	Positive regulation of epithelial cell	101	36	26.26	0.01996
GO:0030216	Keratinocyte differentiation	69	33	17.94	7.60E-05
GO:0051798	Positive regulation of hair follicle development	10	8	2.6	0.00055
GO:0001942	Hair follicle development	67	33	17.42	3.60E-05

Similarly, in H-KER biological processes involved in immune response such as inflammatory response, regulation of T cell chemotaxis, neutrophil chemotaxis as well as processes of hair follicle development and the regulation of the hair follicle development were enriched (Tables 3 and S3). Subsequently, we examined genes belonging to the enriched GO categories.

Table 3

Selected biological processes enriched in H-KER stimulated with allergic cytokine milieu in comparison to non-stimulated H-KER.

GO.ID	Term	Annotated	Significant	Expected	Classic Fisher
GO:0001942	Hair follicle development	69	32	16.22	2.50E-05
GO:0006954	Inflammatory response	304	117	71.45	2.40E-09
GO:0019885	Antigen processing and presentation	8	7	1.88	0.00025
GO:0051798	Positive reg. of hair follicle development	10	8	2.35	0.00026
GO:0051092	Positive reg. of NF-kappaB TF	92	36	21.62	0.00057
GO:0010820	Positive reg. of T cell chemotaxis	7	6	1.65	0.00094
GO:0030593	Neutrophil chemotaxis	47	20	11.05	0.00294
GO:0006955	Immune response	702	227	165	2.40E-08
GO:0071347	Cellular response to interleukin-1	51	25	11.99	0.000061
GO:0022407	Regulation of cell-cell adhesion	250	97	58.76	3.50E-08

a) Transcriptional changes of immune signature. After stimulation with ACM, IBH-KER significantly upregulated expression of genes encoding various interleukins, such as IL31, IL23A, IL36G, IL34, IL6, and IL1A. Additionally, they also significantly upregulated expression of genes encoding different cytokines such as CCL20, CCL27, CCL5 and CCL11 as well as chemokines like CXCL2, CXCL6, CXCL8, CXCL10 and CXCL11. Similarly, H-KER upregulated the same interleukins, and chemokines with exception of CXCL10 and CXCL11 (Table 4A). Interestingly, in IBH-KER the atopic cytokine milieu did not induce expression of epithelial-derived cytokines TSLP, IL25 and IL33, known to play a major role in development of allergic inflammation (Table 4A). In H-KER, only TSLP was significantly upregulated following stimulation with the ACM (log2fold change 0.93) (Table 4A).

Table 4

DEGs are classified by gene families that influence (A) immune responses and (B) epithelial barrier formation and maintenance.

A-B. Cells were analyzed by RNA-sequencing and gene expression was compared between IBH-KER and H-KER stimulated with allergic cytokine milieu (ACM) or unstimulated keratinocytes derived from IBH-affected and H-horses. In (A) representative genes of immune responses and (B) epithelial barrier genes are shown. (Pink = statistically significant upregulation and log2 fold change >1; beige = statistically significant upregulation and log2 fold change <1; dark blue = statistically significant downregulation and log2 fold change >-1; light blue = statistically significant downregulation and log2 fold change <-1; gray = no difference in gene expression; false discovery rate <0.05). Log2 fold changes are noted for all DEGs.

A
Functional group	Gene symbol	IBH-KER	H-KER
Immune signatures		NS vs. ACM	NS vs. ACM
	IL31	5.35	5.44
	IL23A	2.98	2.99
	CXCL8	2.88	2.95
	CXCL10	2.81
	CXCL11	2.70
	IL36G	2.35	2.38
	CCL20	2.08	2.04
	CCL11	1.35	1.98
	CXCL6	1.52	1.91
	IL6	1.37	1.78
	OSMR	1.69	1.60
	IL34	1.27	1.59
	CXCL1	1.22	1.52
	CCL27	1.31	1.44
	IL36RN	1.21	1.23
	CCL5		1.20
	IL1A	1.04	1.07
	TSLP		0.93
	IL31RA	0.45
	IL25
	IL33
B.
Functional group	Gene symbol	IBH-KER	H-KER
Epithelial barrier		NS vs. ACM	NS vs. ACM
	KRT80	-2.14	-2.09
	KRT16	-1.85	-1.75
	SCEL	-1.46	-1.41
	ALOX12N	-1.45	-1.47
	ALOXE3	-1.38	-1.37
	KRT13	-1.35	-1.46
	KRT7	-1.20	-1.15
	FGF9	-1.14	-1.24
	KRT4	-0.75	-1.23
	FGFR2	-1.05	-1.08
	FGF22	-1.03
	KRT6B	-0.76	-0.80
	KRT14	-0.76	-0.74
	FGF1
	KRT8	0.89	1.02
	FGF2	1.09	1.08
	FGFR1

Gene set enrichment analysis (GSEA) using KEGG pathways additionally indicated significant overrepresentation of genes belonging to cytokine-cytokine receptor interaction, chemokine signaling, T cell and B cell receptor signaling pathways and Th1 and Th2 cell differentiation in both IBH-KER and H-KER (S4 and S5 Tables).

Interestingly, IL31 was the highest upregulated gene when comparing ACM-stimulated and non-stimulated cells, in both IBH-KER and H-KER (log2fold change 5.35 and 5.44, respectively). Moreover, IL31 receptor subunit, OSMR, was significantly upregulated in both IBH-KER and H-KER (log2fold change 1.69 and 1.60). IL31RA subunit was significantly upregulated only in IBH-KER, however with lower log2 fold change (log2fold change. = 0.45) (Table 4A). Additionally, JAK-Stat signaling pathway through which IL-31 signals, was also significantly overrepresented in IBH-KER and H-KER stimulated with ACM (S4 and S5 Tables).

b) Transcriptional changes of epithelial barrier. GO analysis indicated that among the enriched biological processes, genes belonging to keratinocyte differentiation and hair follicle development were enriched in keratinocytes stimulated with allergic milieu (Tables 2 and 3). Therefore, we investigated the expression of genes belonging to these processes (Table 4B). Genes involved in keratinocyte differentiation such as SCEL, KRT7, KRT13, KRT16, and KRT80 among others were significantly downregulated. KRT6B and KRT14 were also significantly downregulated, however with lower log2fold change (-0.76 and -0.77, respectively). Only KRT8 expression was upregulated in both IBH-KER and H-KER (log2 fold change = 0.89 and 1.02, respectively). The ACM did not influence the expression of major genes involved in terminal differentiation of keratinocytes like FLG and IVL. Furthermore, genes involved in homeostasis of epithelial lipids, such as ALOXE3 and ALOX12B were significantly downregulated (log2 fold change -1.30 and -1.43, respectively).

In our previous work on transcriptome of lesional skin of IBH-affected horses, we reported significant downregulation of FGFR1 and ligands of FGFR2 in lesional skin of IBH-affected horses [34]. When we investigated the state of FGF receptors and ligands in IBH-KER, we found significant downregulation of FGFR2 and FGF9 as well as FGF22 ligands following exposure to ACM. Expression of FGFR1 was not affected. In H-KER only expression of FGFR2 and FGF9 was significantly downregulated (Table 4B).

Lastly, exposing keratinocytes to the pool of recombinant Culicoides allergens or WBE in combination with ACM resulted only in few significantly upregulated genes compared to stimulation with ACM only (S6, S7, S8 and S9 Tables), which were thus not further analyzed.

Stimulation of primary IBH-KER and H-KER with TLR 1/2-ligand is characterized by transcriptional changes indicative of innate immune responses and impairment in cell proliferation

Lesions of IBH can be further exacerbated by secondary bacterial infections. In order to understand how keratinocytes respond to bacteria, we stimulated primary keratinocytes with the TLR 1/2 synthetic ligand, Pam3CSK4. This stimulation resulted in 206 significantly upregulated and 84 significantly downregulated DEGs in IBH-KER when compared to non-stimulated IBH-KER. In H-KER, 211 genes were significantly upregulated and 169 were significantly downregulated, when compared to non-stimulated H-KER (Fig 5A and 5B).

Fig 5

A-B. Volcano plots of significant DEGs in following comparisons: Non-stimulated (NS) vs.TLR 1/2 ligand (Pam3CSK4) of IBH-KER (A) and H-KER (B).

Non-stimulated and Pam3CSK4-stimulated keratinocytes clustered separately, both in case of IBH-KER and H-KER, as shown in Fig 6. Genes involved in regulation of inflammatory response (TNFAIP3, TNF), NF-κβ (NFKBIZ, KFKB1, NFKBIA) and chemokine signaling (CXCL6, CXCL8) were noticeable among the top 30 DEGs (Fig 6).

Fig 6

A-B. Hierarchical clustering gene expression of top 30 genes of TLR 1/2 ligand (Pam3CSK4)-stimulated and non-stimulated samples from following conditions: IBH-KER (A) and H-KER (B). Lower mean counts are shown in light green and higher mean counts in dark blue.

Interestingly, GO analysis showed that among the top 10 enriched biological processes in IBH-KER and H-KER stimulated with Pam3CSK4 were processes involved in cell cycle, i.e. cell division. Most of the DEGs belonging to processes of cell division were downregulated in both IBH-KER and H-KER (71.4% and 71.43%, respectively). 81% of DEGs belonging to a mitotic cell cycle process in IBH-KER were downregulated and 97.3% of DEGs belonging to the DNA replication process were downregulated, as well (5, 6, S10 and S11 Tables). Furthermore, Kegg pathway based GSEA showed that along with DNA replication and cell cycle pathways, pathways such as Toll like receptor signaling, NOD-like receptor signaling, C-type lectin signaling, Nf-κB signaling pathway were significantly overrepresented in IBH-KER and H-KER stimulated with Pam3CSK4 (S12 and S13 Tables).

Table 5

The 10 most significant biological processes enriched in IBH-KER stimulated with the TLR 1/2 synthetic ligand, Pam3CSK4, in comparison to non-stimulated IBH-KER.

GO-ID	Term	Annotated	Significant	Expected	Classic Fisher
GO:0000278	Mitotic cell cycle	656	231	137.78	2.90E-18
GO:0007059	Chromosome segregation	254	106	53.35	3.30E-14
GO:0000070	Mitotic sister chromatid segregation	127	62	26.67	2.40E-12
GO:0006281	DNA repair	364	127	76.45	3.30E-10
GO:0007093	Mitotic cell cycle checkpoint	100	47	21	5.20E-09
GO:0007052	Mitotic spindle organization	90	40	18.9	4.60E-07
GO:0007094	Mitotic spindle assembly checkpoint	25	16	5.25	4.00E-06
GO:0000724	Double strand break repair via homologous recombination	114	45	23.94	5.00E-06
GO:0006271	DNA strand elongation	16	12	3.36	5.60E-06
GO:0051301	Cell division	211	70	44.32	2.20E-05

Table 6

Top 10 Biological processes enriched in H-KER stimulated with the TLR 1/2 synthetic ligand, Pam3CSK4, in comparison to non-stimulated IBH-KER.

GO.ID	Term	Annotated	Significant	Expected	Classic Fisher
GO:0051301	Cell division	211	77	43.35	4.90E-08
GO:0010950	Positive regulation of endopeptidase activity	106	29	21.78	0.05569
GO:0034501	Protein localization to kinetochore	16	11	3.29	4.20E-05
GO:0031297	Replication fork processing	32	17	6.57	4.60E-05
GO:0006271	DNA strand elongation involved in DNA replication	16	12	3.29	4.40E-06
GO:0000281	Mitotic cytokinesis	47	20	9.66	0.0005
GO:0032922	Circadian regulation of gene expression	45	20	9.24	0.00025
GO:0032508	DNA duplex unwinding	59	27	12.12	1.10E-05
GO:0051988	Regulation of attachment of spindle microtubules to kinetochore	11	7	2.26	0.00231
GO:0034080	CENP-A containing nucleosome assembly	5	5	1.03	0.00036

When we examined genes belonging to these pathways, we found significant upregulation of IL1A, IL23A, IL6 and CSF2 and CSF3 in IBH-KER. Chemokines such as CCL20, CXCL1, CXCL2, CXCL6 and CXCL8 were also significantly upregulated, suggesting strong innate immune activity of keratinocytes (Table 7). We also found significant upregulation of TLR1, TLR6 and TLR10. Log2 fold change of these genes, was however, low (0.48–0.74, respectively). When we further investigated expression of genes belonging to NF-κβ signaling pathway, we found upregulation of NFKB1 and NFKB2, however with lower log2 fold change (0.85 and 0.90) as well as NFKBIA, NFKBI7 and NFKBIE (Table 7). Expression of most of these genes was similar in H-KER, with the exception of IL6 and TLR10; their expression did not differ between stimulated and non-stimulated H-KER (Table 7).

Table 7

DEGs are classified by gene families that influence immune responses in the comparison of IBH-KER and H-KER stimulated with Pam3CSK4 and non-stimulated keratinocytes.

Cell samples were analyzed by RNA-sequencing and gene expressions were compared between Pam3CSK4 stimulated keratinocytes from IBH-affected and H-horses and non-stimulated keratinocytes. Only representative genes are shown. (Pink = statistically significant upregulation and log2 fold change >1; beige = statistically significant upregulation and log2 fold change <1; gray = no difference in gene expression; false discovery rate <0.05). Log2 fold changes are noted for all DEGs.

Functional group	Gene symbol	IBH-KER	H-KER
Immune signatures		NS vs. Pam3CSK4	NS vs. PAm3CSK4
	CSF3	4.63	4.71
	CXCL6	4.26	3.79
	CXCL2	3.19	2.33
	CXCL8	3.10	2.95
	IL23A	2.68	2.96
	IL1A	2.56	2.96
	TNF	2.64	2.95
	CSF2	2.54	2.31
	CXCL1	2.55	2.46
	CCL20	2.41	2.44
	IL36G	2.19	2.23
	NFKBIA	1.94	1.98
	NFKBIZ	1.52	1.48
	NFKBIE	1.22	1.19
	IL6	1.00	0.96
	NFKB2	0.90	0.94
	NFKB1	0.85	0.97
	TRL10	0.78
	TRL6	0.59	0.74
	TLR1	0.48	0.57

Transcriptional differences between IBH-KER and H-KER

In order to investigate whether gene expression in keratinocytes derived from IBH-affected and control horses fundamentally differs, we first compared transcriptomes of non-stimulated keratinocytes derived from IBH-affected and control horses. We found expression of only one gene, CTSL, coding for cathepsin L1 to be significantly upregulated in IBH-KER compared to H-KER (S2 Fig). We furthermore wanted to investigate whether the response of primary equine keratinocytes derived from IBH-affected and control horses differs in response to the stimuli described above. Therefore, we investigated the differences in gene expression between IBH-KER and H-KER stimulated with ACM as well as the TLR 1/2 ligand. Because Culicoides allergen stimulation did not induce any significant changes in comparison to unstimulated keratinocytes (see above), differences in gene expression between IBH-KER and H-KER were not compared.

The transcriptomes of IBH-KER and H-KER stimulated with ACM differed in 23 DEGs (p < 0.05, log2 fold change > 1). Eighteen of those were significantly upregulated and five were significantly downregulated in IBH-KER compared to H-KER (S14 Table). Among upregulated DEGs in IBH-KER were, for example, CXCL10, CXCL11 (p<0.0001, log2 fold change > 1.9), genes involved in chemokine signaling and genes such as IFIH1, IFIH2, IFIT3 and IFI44L, encoding for proteins involved in interferon signaling. Genes such as CH25H and IL34 were significantly downregulated in stimulated IBH-KER (S14 Table), however the FDR for these two genes was close to 5% and they may thus be artifacts.

Transcriptomes of IBH-KER and H-KER stimulated with Pam3CSK4 differed in seven DEGs (p < 0.05, log2fold change >1; S15 Table). Since only a low number of DEGs between the two study groups was found in both keratinocytes stimulated ACM or Pam3CSK4, GO and enrichment analyses were not performed.

Discussion

The role of epithelial barriers in the pathogenesis of Type I hypersensitivities is well-established in human allergy but is only poorly investigated in equine patients. Recently, we reported transcriptome data suggestive of alterations of the epithelial barrier in horses affected with insect bite hypersensitivity. We showed that lesional skin of IBH-affected horses is characterized by transcriptomic evidence of epithelial barrier disruption that is most likely immune mediated. We also found that non-lesional epidermis of IBH-affected horses shows transcriptomic evidence of lipid metabolism disruption and pruritus development which could act as predisposing factor for IBH [34]. In order to investigate a possible role of keratinocytes in the development of Type I hypersensitivities we studied transcriptomes of non-lesional IBH-KER and H-KER and their response to stimulation with Culicoides allergens, ACM and Toll like receptor 1/2 ligand (Pam3CSK4). Unstimulated keratinocytes from IBH-affected and H control horse were very similar at the transcriptional level, as seen by the absence of DEGs, except for CTSL, the gene coding for cathepsin L1. To investigate the response of equine keratinocytes to Culicoides allergens, we stimulated them with a pool of five r-Culicoides allergens as well as whole body extract of C. nubeculosus. The keratinocyte transcriptome did not change under the influence of either the r-allergens or WBE. E.coli expressed allergens are often not suitable for cellular assays [18], because they are often insoluble in inclusion bodies, have to be refolded and lack critical posttranslational modification. Additionally, endotoxin contamination can result in high background cytokine production. For these reasons we used five relevant Culicoides r-allergens produced in P. pastoris. Unfortunately, the allergens expressed in P. pastoris that were available for our study did not include proteases [4]. Recent studies have demonstrated the presence of a much larger number of allergens and proteins in Culicoides saliva, including proteases which are able to disrupt the epithelial barrier and thereby activate keratinocytes [11,16]. Furthermore, it is known that in human allergology, many major allergens are proteases [39-41]. Therefore, we also stimulated primary keratinocytes with WBE of C. nubeculosus. A limitation of our study is that its protease activity was not measured prior to stimulation of keratinocytes. This could account for the lack of stimulation of keratinocytes, which in turn resulted in no DEGs. The use of crude WBE has many limitations, nevertheless, C. nubeculosus WBE have been used with satisfying results for the re-stimulation of PBMCs as well as in basophil activation tests [42-44]. Finally, in vivo, keratinocytes may not only be stimulated by components in the Culicoides saliva but also by the mechanical damage to the skin induced by the bites of Culicoides [11]. Moreover, for studying the baseline response of keratinocytes to Culicoides allergens and to an allergic inflammatory milieu, the keratinocytes used in our study were derived from the non-lesional skin of both IBH-affected and control horses. We collected skin samples or biopsies from the inner thigh, where Culicoides midges do not usually bite. Unfortunately, we could not investigate whether keratinocytes derived from lesional skin respond differently, because in our hands, it was not possible to establish pure keratinocyte culture from lesional skin. Keratinocytes isolated from lesional skin sites might differ from keratinocytes derived from non-lesional sites due to mechanical damage induced by biting of the midges, which may in turn prime the keratinocytes towards a stronger response to allergens due to the damage of epithelial barrier in those sites. Additionally, cells that detach and start proliferating in primary keratinocyte cultures are the basal, proliferative cells. As they proliferate, their differentiation state increases, and they soon stop their replication and die around passage five or six. Therefore, for our experiments we used keratinocytes of passage three that are not fully differentiated yet. This might have also influenced our results, as it was shown that mainly the fully differentiated keratinocytes produce epithelial-derived cytokines such as TSLP [27]. All these factors might account for the lack of transcriptomic differences between Culicoides-stimulated and non-stimulated keratinocytes.

On the other hand, when stimulated with a combination of recombinant equine IL-4 and TNF-α, mimicking an ACM, both IBH-KER and H-KER responded by changes in their transcriptome, suggesting that IBH is not associated with differing responses of IBH-KER or H-KER to an allergic milieu per se, but rather to presence or absence of a local Th2 microenvironment, caused by activation of immune cells such as Th2 lymphocytes, eosinophils and basophils in allergic individuals, which then, secondarily, activates keratinocytes. While an imbalance between the Th2 and T regulatory immune response has been described in IBH, it still remains unknown what are the initiating factors that skew the immune response towards a Th2 response in allergic horses [44,45]. Studies in human patients indicate that the microbiome may play an important role in the development of allergic conditions [46]. However, there is scarce information for the horse and so far no evidence of such effect in IBH [47]. Other factors such as genetic and environmental factors contribute to susceptibility to IBH [4,48]. The age at first exposure to Culicoides allergens also seems to play a crucial role for development of IBH later in life. Horses born in an environment free of Culicoides and exported as adults to Culicoides-rich environments have a much higher prevalence of IBH than horses of the same breed, exported at young age or born in a Culicoides-rich environment [4,48].

Expectedly, after stimulation with ACM keratinocytes upregulated many genes involved in immune responses, including many chemokines and interleukins (Table 4A). Particularly, stimulation with the allergic inflammatory milieu induced a strong upregulation of CCL27, the cutaneous T cell-attracting chemokine which is one of the main cytokines involved in atopic dermatitis (AD) pathogenesis [49,50]. Furthermore, cells also upregulated CCL20 known to be produced in epidermis with impaired permeability (Table 4A). Moreover, CCL20 is also upregulated in human keratinocytes under the influence of TNF-α [51]. Interestingly, due to its involvement in pruritus development, IL-31 has recently been shown to be a therapeutic target in treatment of IBH [52]. In IBH-lesional skin, we recently reported upregulation of both subunits of the IL-31 receptor, IL31RA and OSMR, however, the expression of the cytokine itself was not significantly upregulated. Interestingly, in the present study, upon stimulation of keratinocytes with the ACM, the top significantly upregulated gene in both IBH-KER and H-KER was IL31 (log2 fold change 5.35 and 5.44, respectively). This is the first evidence that equine keratinocytes are capable of producing the Th2 cytokine IL31, following stimulation with allergic micromilieu, and not with TLR 1/2-L. This, however, needs to be further confirmed at the protein level. Unfortunately, antibodies specific for equine IL-31 are not (yet) available. Additionally, IL-31 can also modify the formation of the skin barrier in multiple ways, as demonstrated in human patients. It downregulates the expression of filaggrin, known to be the major protein involved in terminal differentiation of human keratinocytes, weakens the lipid envelope formation and represses enzymes and proteins involved in desmosome formation [53,54]. The role of filaggrin in the pathogenesis of equine IBH has not been studied extensively, but in a transcriptomic study, there was no evidence of an altered expression of filaggrin in lesional IBH skin [55]. In human patients it has now been proposed that IL-31 is a key player in the pathogenesis of AD, and based on our data, IL-31 seems to play an important role in the pathogenesis of IBH [56]. Indeed, upon stimulation with ACM, many genes involved in formation of epithelial barrier were significantly downregulated. SCEL, the gene encoding for sciellin, involved in terminal differentiation of keratinocytes, as well as KRT16, KRT6B and other types of keratin were significantly downregulated, suggesting immune-mediated disruption of the barrier. Interestingly, in our previous study, we found a significant downregulation of SCEL in lesional whole skin of IBH-horses as well as in non-lesional epidermis of IBH-affected horses [34]. Our data thus confirms the importance of sciellin in the epithelial barrier of horses. Furthermore, we found downregulation of FGFR2 and its ligand FGF9 as well as downregulation of FGF1 and FGF22, both ligands of FGFR1. Yang et al. have described a fibroblast growth factor receptor 1 and 2 (fgfr1, fgfr2) knock out mouse model that develops skin lesions similar to those in patients with AD, particularly with regard to the inflammatory infiltrate and the epidermal thickening [57]. Yang et al. attributed the hyperproliferative phenotype to action of IL36B and the S100A8/S100A9 complex. We recently also found transcriptomic evidence of an impairment in FGFR signaling and tight junction disruptions in lesional skin of IBH horses, suggesting that this pathway may indeed play an important role in disruption of epithelial barrier in IBH-affected horses [34]. However, the exact mechanism remains to be elucidated. Taken together, an ACM-induced downregulation of genes involved in epithelial barrier formation suggests the disruption of epithelial barrier by an allergic microenvironment in the horse, similar to human patients [58-60].

IBH-lesional skin is characterized by a strong infiltration with eosinophils [4,61]. However, the mechanism of eosinophil influx still remains largely unknown. In our study, when keratinocytes were stimulated with the ACM, they significantly upregulated the expression of CCL11 in both IBH-KER and H-KER. It has been shown that in human patients IL-4 can induce the production of CCL11 by keratinocytes [62]. Importantly, ICAM1 gene coding for intracellular adhesion molecule 1 which plays a key role in adhesion of eosinophils was significantly upregulated in stimulated equine keratinocytes. CCL5 was significantly upregulated in stimulated H-KER, suggesting a micromilieu-dependent eosinophil homing mechanism.

IBH lesions can be further exacerbated by secondary bacterial infections. In order to investigate how IBH-KER and H-KER respond to bacterial PAMPs, we also stimulated keratinocytes with the toll like receptor 1/2 ligand, Pam3CSK4. In response to this TLR-ligand, both IBH-KER and H-KER showed a strong response inducing TLR and its downstream MyD88 and NF-κβ signaling. Not surprisingly, expression of inflammatory genes was significantly upregulated. Interestingly, genes involved in formation of the epithelial barrier were not affected, unlike following stimulation with the ACM, suggesting that this type of response is specific to the allergic milieu.

Analysis of transcriptional difference between IBH-KER and H-KER stimulated with ACM yielded 23 DEGs and in keratinocytes stimulated with Pam3CSK4 only seven DEGs. However, considering that in our analysis FDR of 5% was taken into account, some of these genes may be artifacts. Furthermore, one healthy horse seems to be an outlier and reacting differently to stimulations (S1 Fig), which also accounts for the difference we saw when comparing IBH-KER and H-KER. This suggests that in this experimental setup, there is no clear transcriptomic difference between IBH-KER and H-KER.

Taken together, our data suggests that equine keratinocytes are, in fact, capable of responding to different stimuli and may play a role in the pathogenesis of IBH, acting as amplifiers of allergic immune reaction through their response to ACM, and thus contributing to the local skin damage in immune-mediator-dependent way. Stimulation with a limited panel of Culicoides r-allergens did not induce a response of keratinocyte. Further studies are needed to assess whether a disruption of the epidermal barrier through mechanical and/or protease induced damage by Culicoides contributes to the initiations of the allergic immune response in IBH or whether skin dendritic cells, innate immune cells and T-cells are the major players.

PCA analysis.

(TIF)

Click here for additional data file.

Volcano plot of significant DEG in the comparison of non-stimulated IBH-KER and H-KER.

(TIF)

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Experimental conditions.

(XLSX)

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Full GO analysis of IBH-KER stimulated with allergic cytokine milieu.

(XLSX)

Click here for additional data file.

Full GO analysis of H-KER stimulated with allergic cytokine milieu.

(XLSX)

Click here for additional data file.

Full KEGG analysis of IBH-KER stimulated with allergic cytokine milieu.

(XLSX)

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Full KEGG analysis of H-KER stimulated with allergic cytokine milieu.

(XLSX)

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DEGs from IBH-KER stimulated with allergic cytokine milieu versus IBH-KER stimulated with allergic cytokine milieu in combination with Culicoides recombinant proteins.

(XLSX)

Click here for additional data file.

DEGs from H-KER stimulated with allergic cytokine milieu versus H-KER stimulated with allergic cytokine milieu in combination with Culicoides recombinant proteins.

(XLSX)

Click here for additional data file.

DEGs from H-KER stimulated with allergic cytokine milieu versus H-KER stimulated with allergic cytokine milieu in combination with Culicoides whole body extract.

(XLSX)

Click here for additional data file.

DEGs from IBH-KER stimulated with allergic cytokine milieu versus IBH-KER stimulated with allergic cytokine milieu in combination with Culicoides whole body extract.

(XLSX)

Click here for additional data file.

Full GO analysis of IBH-KER stim with Pam3CSK4.

(XLSX)

Click here for additional data file.

Full GO analysis of H-KER stim with Pam3CSK4.

(XLSX)

Click here for additional data file.

Full KEGG analysis of IBH-KER stim with Pam3CSK4.

(XLSX)

Click here for additional data file.

Full KEGG analysis of H-KER stim with Pam3CSK4.

(XLSX)

Click here for additional data file.

DEGs from IBH-KER + allergic cytokine milieu versus H-KER + allergic cytokine milieu.

(XLSX)

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DE Gs from IBH-KER + Pam3CSK4 versus H-KER + Pam3CSK4.

(XLSX)

Click here for additional data file.

31 Aug 2021

PONE-D-21-13837

Equine keratinocytes in the pathogenesis of insect bite hypersensitivity: just another brick in the wall?

PLOS ONE

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Reviewer #1: The papers describes the transcriptome of keratinocytes isolated from IBH- or healthy horses, non-lesional skin. The investigations and descriptions have been performed in depth, and presented in a clear manner.

The limitations of the study (no parts from lesional skin, missing recombinant allergens like proteases, no enzymatic activity testing of used WBE etc.) have been listed and discussed.

Major point:

* due to missing info for lesional skin and important allergens from Cul. saliva, and also in light of no differences between IBH and healthy horses as well as in practice not being able perform a longitudinal study (horses before and after IBH-development), I would tune down all the statements suggesting kerationcytes as being responsible in IBH development (e.g. line 25)!

* include in discussion the contribution of genetics/breed, environment, feed, hygiene treatment etc. of animals in IBH development

* as an ACM alters transcriptome in both, IBH and healthy horses, discuss a) where that ACM might come from in a natural setting and what might be different between allergy-susceptible and healthy animals; and b) if both IBH and healthy keratinocytes react to ACM, why don't healthy develop allergies (i.e. don't they ever have a situation/setting where ACM is around, or do they have an efficient regulatory response?).

Minors:

* short title should be short

* line 37: introduce abbreviations of IHB-KER/H-KER in line 26

* line 38: name the single gene that differed

* line 58: "...due to strong pruritus and inflicted self-trauma"

* line 72: "...have been shown..."; add why E.coli expressed proteins are of limited use

* line 76: complete with "The binding of IgE to the FceRI and crosslinking of bound IgE with allergen activates..."

* line 96: instead of natural model better use "a valuable source of information from horses with spontaneously ocuring disease for the role of kerationcytes in allergic skin diseases also for other species."

* line 99: include reference

* line 108: Pam3CSK4: add that it is a synthetic ligand

* Methods:

- include time-point of skin sampling and medium as well as temperature for transport and storage after slaughtering;

- include stimulation conditions (conc. of stimulations, incubation time, temperature etc.); Table S1 is not sufficient

- line 145: which staining?

- line 163: 70 or 80% confluence (stated at different places in MS)

- throughout MS, take care to put space between "pvalues"

- table 4 and 5 need different titles/sub-titles

* line 411: state in paragraph again that Cul. allergens did induce no changes, therefore were not compared between KER and H horses

* line 422: 23 DEGs were later discussed as artefacts - insert that info already in results section!

* line 453: "either" "or" instead of neither/nor

* 454: "had been proven"

* line 457: again explain why E.coli expr. proteins are not suitable

* line 459: insert "relevant" allergens

* lines 461-463: change order of sentences

* line 488: mimicking an allergic cytokine milieu with IL-4 and TNF-alpha: discuss again where that would come from in IBH horses vs. healthy

* line 500: "interestingly, in the present study upon stimulation..."

* line 515: should be sciellin

* Figures: please put in addition to figure legend the labels of the different figure parts also directly in figure for better legibility, e.g. left panels title IBH-KER vs. right panel titles IBH-H, and rec. allergens vs. WBE vs. ACM in Fig.2; like done in Fig. 4

* Discussion: is Filaggrin known to be important in horses as well (e.g. null-mutations)?

Reviewer #2: Dear Authors,

The submitted manuscript describes a role for equine keratinocytes in the mechanism of allergy (specifically equine IBH) by acting as bystander responders to allergy-associated cytokines. This provides a link between immune cells and epidermal cells in amplifying an allergic response. Please see below my review comments on your manuscript.

MAJOR COMMENTS

1. Please add more background on the selection of IL-4 and TNF-a, but not others, and what is known about these two cytokines in IBH pathogenesis. Why were other Th2 cytokines, such as IL-5 and IL-13, not included? How did you determine the concentrations of the ACM? How did you determine the concentrations of the r-allergen, WBE and TLR ligand stimulation conditions?

2. Please clarify the stimulation conditions in the methods. Line 165 should say “…WBE OR toll like…”. Figure 1 suggests that some samples were co-stimulated with r-allergens or WBE and also with ACM. However, those data are not presented. Either show the data from co-stimulations or clarify Figure 1.

3. Does pre-exposing KER to r-allergens/WBE change the transcriptional response after subsequent ACM stimulation? In other words, is there an additive effect of allergen and cytokine on the KER response?

4. Add a figure validating the purity and identity of keratinocyte cultures.

5. Figures 4 and 7 are challenging to interpret with many different color codes. I suggest adding FDR/p(adj) values as well. And/or change the order of genes in descending Log2 fold change so each color-coded category is grouped together. Graphical representation of these data would also be useful.

6. Please comment in the discussion how keratinocytes in lesional skin of IBH horses may differ from the keratinocyte samples compared in this study.

MINOR COMMENTS

1. Should line 187 say 17-31 MIL 2x50bp? Correct typo if it is one.

2. The figure titles and captions need to be improved. The first sentence should summarize the point of the figure (ex: in Fig 2 this could just be “Volcano plots of significant DEGs in different comparisons”), and the following unbolded sentences should describe the different subparts of the figure, describing sample types, color coding, etc. Currently, the figure titles are what should be the caption.

3. In Figures 2+5, it would be beneficial to label the genes of interest that are highlighted in Figures 4+7.

4. Line 452-453 is a double negative. Should be “did not change…either of the r-allergens….”

5. Line 460: activate

6. Line 556: artifacts

7. Line 556: outlier

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Reviewer #1: No

Reviewer #2: No

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4 Feb 2022

Dear Editor, dear Reviewers,

We thank the reviewers and editor for their useful comments. We have corrected the manuscript accordingly. Below please find our response to the reviewer with the corresponding line numbers in the revised manuscript marked in red.

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Thank you for the comment, we think that the manuscript meets PLOS ONE's style requirements.

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please ensure that you have specified (1) whether consent was informed and (2) what type you obtained (for instance, written or verbal). If the need for consent was waived by the ethics committee, please include this information.

The samples were all taken from slaughter horses, except for one horse, for which written consent was obtained. This information is now provided in lines 138-139.

3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

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4. Thank you for stating the following in the Acknowledgments Section of your manuscript:

“We thank Jelena Mirkovitch and Shui Chu Ling for their expert laboratory assistance. Arthropods used in this study were provided by the Pirbright Institute under UK under grant code BBS/E/I/00007039 awarded to Dr Simon Carpenter as part of funding received from the Biotechnology and Biological Science Research Council (UKRI).”

The Funding information has been removed from the acknowledgments.

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

Please update the Funding Statement as completed in red below:

“This work was supported by the Swiss National Science Foundation grant no. 310030-160196/1. This SNF grant was awarded to E.M. http://www.snf.ch/. Arthropods used in this study were provided by the Pirbright Institute under UK under grant code BBS/E/I/00007039 awarded to Dr Simon Carpenter as part of funding received from the Biotechnology and Biological Science Research Council (UKRI).

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript”

5. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

Repository information is provided in lines 240 to 241: “The datasets generated during the current study are available in the ENA repository via accession numbers PRJEB37568 “.

6. We note that you have referenced (five r-allergens that had proven to induce sulfidoleukotriene release in IBH-affected but not in healthy horses in a basophil activation test) which has currently not yet been accepted for publication. Please remove this from your References and amend this to state in the body of your manuscript [Unpublished]”) as detailed online in our guide for authors data as supplementary material

http://journals.plos.org/plosone/s/submission-guidelines#loc-reference-style

We have removed this sentence from the text as we cannot provide the unpublished data. We would like to include this data in a later manuscript specific for the study that we had mentioned.

We have one new co-author, who did the new figure 1.

________________________________________

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The papers describes the transcriptome of keratinocytes isolated from IBH- or healthy horses, non-lesional skin. The investigations and descriptions have been performed in depth, and presented in a clear manner.

The limitations of the study (no parts from lesional skin, missing recombinant allergens like proteases, no enzymatic activity testing of used WBE etc.) have been listed and discussed.

Major point:

* due to missing info for lesional skin and important allergens from Cul. saliva, and also in light of no differences between IBH and healthy horses as well as in practice not being able perform a longitudinal study (horses before and after IBH-development), I would tune down all the statements suggesting kerationcytes as being responsible in IBH development (e.g. line 25)!

Thank you for your comment.

Line 43: We have written that equine keratinocytes have the ability of eliciting differing responses to different stimuli and may possibly play a role in the pathogenesis of IBH. In the next lanes we explain that they are not the primary responders but that they can amplify the allergic immune response by their activation mediated by allergic inflammatory cytokines, such as IL-4 and TFN- a.

Furthermore, in the discussion, lines 621-628 we address this matter and further elaborate it.

* include in discussion the contribution of genetics/breed, environment, feed, hygiene treatment etc. of animals in IBH development

Thank you for the comment. This is now mentioned in lines 536-544.

* as an ACM alters transcriptome in both, IBH and healthy horses, discuss a) where that ACM might come from in a natural setting and what might be different between allergy-susceptible and healthy animals; and b) if both IBH and healthy keratinocytes react to ACM, why don't healthy develop allergies (i.e. don't they ever have a situation/setting where ACM is around, or do they have an efficient regulatory response?) ;

Thank you for the insight. This is now added in the following lines: 92-97 and 529-536.

Minors:

Thank you for the minor revisions. They are corrected as follows:

* short title should be short : Corrected in line 17

* line 37: introduce abbreviations of IHB-KER/H-KER in line 26; line 27

* line 38: name the single gene that differed ; line 39

* line 58: "...due to strong pruritus and inflicted self-trauma" ; line 59

* line 72: "...have been shown..."; add why E.coli expressed proteins are of limited use ;

This is now clarified in lines 72-74: … such as endotoxin contamination and lack of post-translational modification of the protein, leading to unspecific stimulations or lack of response, respectively, and thus limiting their use for cellular in vitro assays [19].

* line 76: complete with "The binding of IgE to the FceRI and crosslinking of bound IgE with allergen activates..." ;added to line 79

* line 96: instead of natural model better use "a valuable source of information from horses with spontaneously ocuring disease for the role of kerationcytes in allergic skin diseases also for other species." ; line 106-108

* line 99: include reference, line 110

* line 108: Pam3CSK4: add that it is a synthetic ligand ; line 118

* Methods:

- include time-point of skin sampling and medium as well as temperature for transport and storage after slaughtering; line 136-139

- include stimulation conditions (conc. of stimulations, incubation time, temperature etc.); Table S1 is not sufficient; Table S1 edited

- line 145: which staining?; line 159

- line 163: 70 or 80% confluence (stated at different places in MS); lines 152, 158

- throughout MS, take care to put space between "pvalues"; corrected throughout the manuscript

- table 4 and 5 need different titles/sub-titles

Thank you! The titles of table 4 and 5 have been corrected

* line 411: state in paragraph again that Cul. allergens did induce no changes, therefore were not compared between KER and H horses;

This information is now added in lines 453-455.

* line 422: 23 DEGs were later discussed as artefacts. This was not well formulated and has been adapted (line 615) - insert that info already in results section!; has been added: lines 463-464

* line 453: "either" "or" instead of neither/nor ; line 489

* 454: "had been proven" ; rewritten in line 491

* line 457: again explain why E.coli expr. proteins are not suitable ; line 490-493

* line 459: insert "relevant" allergens ; line 493

* lines 461-463: change order of sentences ; line 495-498

* line 488: mimicking an allergic cytokine milieu with IL-4 and TNF-alpha: discuss again where that would come from in IBH horses vs. healthy ; line 531-533

* line 500: "interestingly, in the present study upon stimulation..." line 556

* line 515: should be sciellin; line 574

* Figures: please put in addition to figure legend the labels of the different figure parts also directly in figure for better legibility, e.g. left panels title IBH-KER vs. right panel titles IBH-H, and rec. allergens vs. WBE vs. ACM in Fig.2; like done in Fig. 4;

This was now corrected in figures 3A-F, 4A-B, 6 A-B, 7 A-B

* Discussion: is Filaggrin known to be important in horses as well (e.g. null-mutations)?; no there is no evidence so far, line 567-569

Reviewer #2: Dear Authors,

The submitted manuscript describes a role for equine keratinocytes in the mechanism of allergy (specifically equine IBH) by acting as bystander responders to allergy-associated cytokines. This provides a link between immune cells and epidermal cells in amplifying an allergic response. Please see below my review comments on your manuscript.

MAJOR COMMENTS

1. Please add more background on the selection of IL-4 and TNF-a, but not others, and what is known about these two cytokines in IBH pathogenesis. Why were other Th2 cytokines, such as IL-5 and IL-13, not included? How did you determine the concentrations of the ACM?

How did you determine the concentrations of the r-allergen, WBE and TLR ligand stimulation conditions?;

Thank you for your comment. This is added in lines 92-97 and 194-196. At the time of the experimental part of the study, equine recombinant IL-5 and IL-13 were not, unlike nowadays, available. Additionally, we wanted to focus more on IL-4, being shown to be upregulated in the skin of IBH-affected horses. However, considering the novel data from human medicine showing that IL-13 seems to be the key player in the peripheral sights (e.g. skin) of atopic individuals, and our data from sequencing full lesional skin of IBH-horses that showed upregulation of IL13, it would have been very interesting to have a group of keratinocytes stimulated with IL-13 alone or in combination with IL-4 and TNF-alpha.

2. Please clarify the stimulation conditions in the methods. Line 165 should say “…WBE OR toll like…”.

Thank you has been modified. Line 189

Figure 1 suggests that some samples were co-stimulated with r-allergens or WBE and also with ACM. However, those data are not presented. Either show the data from co-stimulations or clarify Figure 1. ;

Thank you for the valuable insight. Four supplementary tables (Table S6, -S7, -S8 and -S9) were added, and text in lines 376-379 was accordingly added.

3. Does pre-exposing KER to r-allergens/WBE change the transcriptional response after subsequent ACM stimulation? In other words, is there an additive effect of allergen and cytokine on the KER response?

Thank you for the comment. This is now added in lines 376-379.

4. Add a figure validating the purity and identity of keratinocyte cultures.

Thank you for comment. A Figure was added as Figure 1.

5. Figures 4 and 7 are challenging to interpret with many different color codes. I suggest adding FDR/p(adj) values as well. And/or change the order of genes in descending Log2 fold change so each color-coded category is grouped together. Graphical representation of these data would also be useful.

Thank you for the comment. Figures were changed so that change of the order of genes is in descending Log2 fold change and therefore each color-coded category is grouped together (now Figure 5 A-B and Figure 8).

6. Please comment in the discussion how keratinocytes in lesional skin of IBH horses may differ from the keratinocyte samples compared in this study.

Thank you for your insight. This is added in lines 514-518.

MINOR COMMENTS

Thank you very much for the minor comments, as well!

1. Should line 187 say 17-31 MIL 2x50bp? Correct typo if it is one. ; line 213

2. The figure titles and captions need to be improved. The first sentence should summarize the point of the figure (ex: in Fig 2 this could just be “Volcano plots of significant DEGs in different comparisons”), and the following unbolded sentences should describe the different subparts of the figure, describing sample types, color coding, etc. Currently, the figure titles are what should be the caption.

This was changed throughout the manuscript.

3. In Figures 2+5, it would be beneficial to label the genes of interest that are highlighted in Figures 4+7

Unfortunately these figures are produced by a software where the labeling of genes is not possible.

4. Line 452-453 is a double negative. Should be “did not change…either of the r-allergens….”; line 489

5. Line 460: activate; rewritten, now line 498

6. Line 556: artifacts ; line 615

7. Line 556: outlier ; line 616

________________________________________

Submitted filename: Response to reviewers.docx

Click here for additional data file.

15 Feb 2022

15 Mar 2022

Dear Editor, dear Reviewers,

Thank you for you useful comments.

We have corrected the manuscript according to the reviewer's comments. Changes are marked in light blue in the revised manuscript with track changes.

There is just one comment (major revision no 3) that we cannot address. We think that validation of the RNAseq results with qPCR analysis of lesional and/or nonlesional skin is beyond the scope of this manuscript, as we have studied the response of primary keratinocyte cultures derived from skin of IBH-affected and healthy horses following in vitro stimulations. Furthermore, a recent study showed by qPCR that IL-31 was detectable exclusively in the lesional skin of IBH-affected horses, but not detectable in the skin of healthy horses (Olomski et al., 2020). This study is cited in our manuscript (Ref no 37). Additionally, we are not able to perform further experiment because the first author of our paper is no longer working in our group and our technician is unfortunately on a longer-term sick leave. Therefore, at the moment, it would be impossible for us to perform qPCRs or other experiments. We thus hope that our manuscript will be acceptable for publication without these additional experiments.

We have made no changes to the financial and we have made no changes to the references in this revision of the manuscript.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

________________________________________

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: All comments have been adequately addressed, points that could not be answered or investigated have been sufficiently discussed as limitations of the study.

Reviewer #2: The authors have addressed many of the prior concerns and revisions to their manuscript describing the role of equine keratinocytes in insect bite hypersensitivity. Thank you. They also describe the importance of cytokine signaling in developing and exacerbating the allergic response. Most of my revisions are minor grammatical corrections. I do have some major revisions regarding that should be addressed before acceptance. Please see my recommendations below.

Major revisions

1) Lines 42-44 and 620-621. These two sentences need to be changed to better describe the identity of keratinocytes. While the data support that keratinocytes are involved in the first line of immune defense, it is incorrect to state that they are “bona fide innate immune cells”. Expression of immune related genes shows that they are involved in the immune response, but calling them “innate immune cells” would mean that they are hematopoietically derived, which is not shown with the data and cannot be based on gene expression alone. Further characterization of cell ontogeny would be needed to call them this. Also, epidermal cells are involved in the immune response even though they are not immune cells.

Please modify these sentences in the abstract and discussion/conclusion to better describe the identity and role of keratinocytes in the immune response. Please remove “bona fide innate immune cells”.

Here is an example for the abstract: “Our data suggests that equine keratinocytes contribute to the innate immune response and are able to elicit responses to different stimuli, possibly playing a role in the pathogenesis of IBH.”

Thank you very much for your comprehensive insight. The changes were adapted in the lines 45-47 and 617-618.

2) Figures 5 and 8 are both tables and I recommend that they be turned into tables instead of figures. Below are a few specific changes that should be made:

Thank you for your suggestion. The figures are now turned into tables.

-Line 332: Please fix the title to read more clearly. I recommend this, or similar:

“DEGs are classified by gene families that influence (A) the immune response and (B) epithelial barrier formation and maintenance.”

Thank you very much. Title is now fixed in 332-333.

-Line 334-340 and 438-441: Please add to the legend more details about the experiment that was done. In the first sentence, please add that the cell samples were analyzed by RNA-seq and that gene expression was compared between IBH-KER and H-KER. This will be redundant with the figure/table text but will greatly enhance clarity and accurate interpretation.

Thank you. The suggested changes are now implemented in lines 333-336 and 444--448.

The line 332 and 334-3340 and

-Line 334-340: I recommend re-stating in the legend (not just the title) that (A) shows upregulated immune response genes and (B) shows upregulated epithelial barrier genes. Alternatively, you could combine the table into one table instead of two.

Thank you! The changes are implemented in the lines 336-337.

3) It would be helpful to validate the RNAseq results with qPCR analysis of lesional and/or nonlesional skin. This could be shown in new Figures 5+8 if the current figures become Tables. Please consider adding a graph that shows the gene expression of key genes (IL31, KRT80, etc) in the different conditions.

In this manuscript we have investigated the response of primary keratinocytes derived from skin of IBH-affected and healthy horses following in vitro stimulations. We find that validating our findings in lesional and nonlesional skin is thus beyond the scope of the study presented here. Furthermore, a recent study showed by qPCR that IL-31 was detectable exclusively in the lesional skin of IBH-affected horses, but not detectable in the skin of healthy horses (Olomski et al., 2020). This study is cited in our manuscript (Ref no 37).

Additionally, we are not able to perform further experiment because the first author of our paper is no longer working in our group and our technician is unfortunately on a longer-term sick leave. Therefore, at the moment, it would be impossible for us to perform qPCRs or other experiments.

Minor revisions

Thank you very much. All the minor revisions are implemented in the following lines:

Line 92: Add period/punctuation after milieu. Line 92.

Line 93: Introduce ACM abbreviation here and use throughout. Line 93 and all throughout manuscript.

Line 101: Fix grammar to say “consisting of A combination of recombinant...”. Line 101.

Line 102, 104: Capitalize “Type I...”. Please make sure this is correctly capitalized throughout manuscript. Lines 102, 104 and all throughout manuscript.

Tables 2+3: Some of the term names are cut off. Please adjust so that they fit in the table, or add full term name as a table footnote. Implemented in both tables.

Lines 320 + 355: Section titles are both listed as “A)”. Please fix to say “a” and “b”, or remove lettering list.

Implemented in lines 320 (a) and 361 (b).

Lines 324-325: Chemokine genes do not have a dash in them, according to nomenclature rules. Please fix here and also make sure they are correctly named throughout. For example, chemokine gene names should read as “CCL20”. Lines 324-325 and throughout manuscript.

Line 327: Please add “, the” after IBH-KER so that the sentence here is more clear:

“Interestingly, in IBH-KER, the atopic cytokine...” Line 327.

Line 409: Fix grammar to say “belonging to the DNA replication process...” Line 415.

Submitted filename: Response to reviewers-2nd revision.docx

Click here for additional data file.

18 Mar 2022

Equine keratinocytes in the pathogenesis of insect bite hypersensitivity: just another brick in the wall?

PONE-D-21-13837R2

Dear Dr. Marti,

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27 Jun 2022

PONE-D-21-13837R2

Equine keratinocytes in the pathogenesis of insect bite hypersensitivity: just another brick in the wall?

Dear Dr. Marti:

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