Cytokeratin expression and distribution pattern of epithelioid macrophages in granulomatous lesions of animals with different pathological forms of bovine paratuberculosis: cytokeratin as a biomarker of resilience.

Cytokeratin expression and distribution pattern of epithelioid macrophages in granulomatous lesions of animals with different pathological forms of bovine paratuberculosis: cytokeratin as a biomarker of resilience. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cytokeratin expression and distribution pattern of epithelioid macrophages in granulomatous lesions of animals with different pathological forms of bovine paratuberculosis: cytokeratin as a biomarker of resilience. Alejandra Isabel Navarro León, Marta Muñoz, Cristina Blanco-Vázquez, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3982091/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract A recent genome-wide association study identified 92 genetic variants in cattle with paratuberculosis (PTB)-associated multifocal lesions. Pathway analysis with the identified candidate genes revealed a significant enrichment of the keratinization (KRT) pathway in those animals. To confirm, at the protein level, this enrichment the number and distribution pattern of cytokeratin (CK)-expressing cells in granulomas of distal jejunum (DJE) and jejunal lymph nodes (JELN) of animals with different PTB-associated lesions (focal, multifocal and diffuse) and in control animals without lesions was determined by quantitative double-immunohistochemical analysis using Iba1 (ionized calcium-binding adapter molecule-1) and CK as specific markers of macrophages and epithelial cells, respectively. Animals with multifocal lesions showed the highest numbers of double-Iba1/CK positive cells (epithelioid macrophages (EMs)) showing significant differences with focal, diffuse and control animals in JELN and higher numbers of single-CK expressing cells in JELN and DJE. Two distribution patterns of the EMs in the granulomas were observed. In focal and multifocal animals EMs were surrounding the granuloma forming a barrier crucial to control Map infection while in animals with diffuse lesions (with significantly higher infection scores) EMs were throughout all the extension of the granuloma. Multifocal animals might be resilient to the disease as they control the shift from subclinical to the clinical through formation of ordered granulomas where EMs have a relevant role preventing Map dissemination and maintaining tissue integrity. Since CK expression was enriched in cattle with multifocal lesions, it could be considered as a potential biomarker of PTB resilience. paratuberculosis cytokeratin epithelioid macrophages granuloma resilience Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Bovine paratuberculosis (PTB) is a chronic granulomatous enteritis caused by Mycobacterium avium subsp. paratuberculosis (Map) responsible of important economic losses in the dairy industry. Map has been associated with several inflammatory and autoimmune diseases in humans such as Crohn’s disease [ 1 ], rheumatoid arthritis [ 2 ], type I diabetes [ 3 ], or multiple sclerosis [ 4 ]. PTB is listed by the World Organization for Animal Health (OIE) as a noticeable disease [ 5 ] due to its potential public health implications and its direct effects on animal health, economic losses, and livestock trade. Depending on the severity of the clinical signs observed, the potential for shedding bacteria into the environment, and the ease with which the disease may be detected by current laboratory methods, four stages of PTB are defined [ 6 ]: silent, subclinical, clinical, and advanced clinical. In Map infected animals, a variety of granulomatous lesions associated with the different clinical stages of the disease can be detected, which have been classified in three main types: focal, multifocal, and diffuse [ 7 ]. Focal lesions consist of small scattered and well-demarcated granulomas composed by macrophages and few Langhans giant cells, mainly located in the jejunal (JELN) and ileal lymph nodes (ILN) and not affecting the intestinal lamina propria. Multifocal lesions consist of numerous well-demarcated granulomas in the intestinal lymphoid tissues and in the intestinal lamina propria. Diffuse lesions are characterized by extensive severe inflammatory infiltrate with granulomas in the intestinal lymphoid tissues and lamina propria, that markedly alter the normal histological structure of the intestine including submucosa, resulting in a clear merger of the villi. According to the inflammatory cell type present in the infiltrate and the number of acid-fast bacilli, diffuse lesions are further subdivided into diffuse lymphoplasmacytic paucibacillary, diffuse intermediate, and diffuse histiocytic multibacillary lesions [ 8 ]. Map infection produces a chronic and granulomatous inflammatory response characterized by the presence of granulomas, host-derived immune structures that consist of mononuclear cells that differentiate into macrophages, epithelioid cells, and multinucleated giant cells. Macrophages forming the granulomas are at different activation stages, which may or may not be associated with other inflammatory cell types [ 9 – 11 ]. Macrophages are phagocytic cells that play a central role in the defence of the host, as part of the host's innate immune response to Map infection [ 12 ]. Map is engulfed by intestinal macrophages as part of the initial response to infection, however, Map can disrupt normal macrophage functions and avoid destruction by preventing phagolysosome fusion, acidification, and activation [ 9 , 12 – 14 ]. Map can hide within infected macrophages during the long subclinical stage of PTB. Only a small percentage of the cattle exposed to Map developed clinical [ 13 , 15 ]. There are other factors such as host genetics, environmental conditions and the infectious dose that also determine the animal’s ability to control MAP infection. PTB progression results in various changes of the macrophages within the granuloma [ 16 ], resulting in a range of histological appearances [ 7 , 17 ]. One of these changes is the epithelioid transformation of macrophages which produces cells with flattened shape, ovoid nuclei, and membranes that interdigitate with adjacent cells leading to the denomination epithelioid macrophages (EMs), also known as epithelioid cells. Although these cells are macrophage-derived, EMs are regarded as a very specialized type of mononuclear phagocyte immobilized in the granuloma, whose function has changed from phagocytosis to extracellular secretion [ 10 ]. Epithelial macrophages express at least one canonical epithelial marker. Cytokeratin (CK), a cytoskeletal protein highly specific of epithelial cells [ 18 ], has been previously used as a marker of epithelioid macrophages during the evolution of chronic granulomatous inflammation in a teleost fish model (pacus, Piaractus mesopatamicus ) induced by Bacillus Calmette-Guerin (BCG) demonstrating that macrophages with phagocytic activity became transformed into epithelioid cells with secretory activity [ 19 ]. CK has also been used as an epithelioid cell marker in the ulcerative mycosis granulomas of Atlantic menhaden [ 20 ]. The associations between host genetics and the different PTB-associated lesions have been studied [ 21 ] identifying 192 and 92 single nucleotide polymorphisms (SNPs) that define 13 and 9 quantitative trait loci (QLTs) associated with multifocal and diffuse lesions, respectively. They suggest that distinct genetic variants might control the multifocal and diffuse lesions as no overlap was seen in the SNPs associated with each type of these lesions which could represent divergent disease outcomes. Pathway analysis with candidate genes overlapping the identified QLTs revealed a significant enrichment of the keratinization pathway in the animals with multifocal lesions. Gene members of the keratin (KRT) family such as KRT5, KRT7, KRT72, KRT73, KRT74, KRT75, KRT80, KRT81, and KRT83 were associated with the multifocal lesions. KRTs belong to the largest subgroup of intermediate filament (IF) family of cytoskeletal proteins and represent the most abundant proteins in epithelial cells, where they play a major role protecting epithelial cells from cell death, repairing cells and maintaining the stability and integrity of the gastrointestinal epithelium. They carry out other functions being, for example, responsible for the response and adaptation to various stresses, as conveyed by the broad array of crippling clinical disorders caused by inherited mutations in KRT coding sequences [ 22 ]. For instance, expression of KRT5 in distal airway stem cells is essential for lung regeneration after H1N1 influenza virus infection [ 23 ], KRT7 may play a role in the histogenesis of small intestinal carcinoma associated with Crohn's disease [ 24 ] and the KRT75 gene has been shown to be a candidate gene associated with heat stress adaptation in Chinese cattle [ 25 ]. Thus, CK expression could contribute to the containment of Map infection in several ways. Canive et al. (2021) [ 21 ] hypothesized that the overexpression of CK in animals with multifocal lesions could provide tissue resilience through formation of epithelioid granulomas. The function of granuloma and the consequences of epithelioid transformation in granulomas caused by mycobacterial infections are not well understood. Thus, the starting hypothesis of the work presented here is that genetic variants of the keratinization pathway may predispose cattle to develop multifocal lesions able to prevent Map dissemination and limit tissue damage through formation of ordered granulomas where EMs, secretory cells that express CK, have a relevant role in controlling infection. In this context, the aim of the present study was to confirm, at the protein level, the enrichment of the keratinization pathway in cattle with multifocal lesions, and to investigate whether the presence of multifocally distributed epithelioid granulomas leads to the control of Map infection. For this purpose, the number and distribution pattern of EMs in distal jejunum (DJE) and JELN granulomas of animals with different pathological forms associated with PTB (focal, multifocal, diffuse intermediate, and diffuse multibacillary) and in negative control animals without lesions was investigated through quantitative double-immunohistochemical (D-IHC) analysis (using ionized calcium-binding adapter molecule-1 (Iba1) and CK as markers of macrophages and epithelial cells, respectively. Materials and Methods Animals and Samples Cattle were selected from two farms located in the Principality of Asturias (Northwest of Spain). A total of 32 Holstein Friesian cows (0.81-10.39 years) were used for this study. Samples of blood, faeces and tissues were collected from all the animals. Blood samples were taken in serum clot activator Vacutainer® tubes (Vacuette, Kremsmunster, Austria) from the coccygeal vein and then transported to our laboratory at room temperature (RT). Serum was separated by centrifugation (2500 x g for 20 min at RT), Samples of faeces were collected, aliquoted and then stored at −20°C for subsequent ELISA analysis. Tissue samples (DJE, ileocecal valve (ICV), and ILN and JELN) were collected from the slaughtered animals in situ at the local abattoir after evisceration and they were used for histological classification and D-IHC (DJE and JELN). The Map infection status of the 32 animals used in this study was determined by histopathology, specific antibody serum ELISA test (IDEXX, Montpellier, France), bacteriological culture, and specific real-time PCR of tissues and faeces, following the procedures previously described [26]. As an indirect estimation of the amount of Map present in each histopathological group (our gold standard) we created an infection score assuming that the amount of Map present in an animal is related to the positivity found in ZN, ELISA, PCR and the bacteriological culture. The mean infection score of each histopathological group was calculated as the sum of the total number of positive diagnosis test (counting 1 for each positive, 0.5 for each non-conclusive result and 0 for each negative result) plus the total number of ZN positive results (1 if there is one + in the ZN result, 2 for two ++ and 3 for +++) split by the total number of animals in the group. Tissue preparation and histopathological classification of animals Tissue samples were taken and processed using standard procedures. Samples were fixed in 10% neutral buffered formalin, sliced, and embedded in paraffin blocks. Tissue sections (4 µm) were cut and placed on microscope slides (Superfrost Plus, Menzel GmbH, Braunschweig, Germany), and dried at 37 ° C for 24 h. Afterwards, tissue sections were stained by hematoxylin-eosin (HE) and Ziehl-Neelsen (ZN) to evaluate lesions and confirm the presence of acid-fast bacteria. Slices were analysed using an Olympus BH-2 light microscope (Olympus, Tokyo, Japan). Pathological lesions associated with bovine PTB were classified according to Gonzalez et al. (2005). The classification was carried out by examining four complementary target sections of the gut tissue (DJE, ICV, ILN and JELN). Once examined the four sections for all the animals (focal, multifocal, and diffuse) included in the study, DJE and JELN were selected to carry out the D-IHC as these two sections showed overall more abundant granulomas. In multifocal animals, the main target group under study, most granulomas were present in DJE (the number of granulomas in ICV was lower) and JELN. Focal animals did not often present granulomas in DJE lymphoid tissue, so examination of JELN sections was important to the experiment. Diffuse lesions were observed in the four sections. Double-immunohistochemistry (D-IHC) For quantification of the number of CK-positive macrophages (EMs) within granulomas of animals with different pathological forms associated with PTB (focal, multifocal, diffuse intermediate, and diffuse multibacillary) and in negative control animals without lesions the number of cells expressing CK and Iba1 within the DJE and JELN of each animal was investigated by sequential double-immunohistochemistry to detect two different antigens Iba1 and CK in tissue samples (Additional File 1). Formalin-fixed paraffin-embedded DJE, and JELN samples were cut into 3-µm sections and placed on microscope slides (Superfrost Plus, Menzel GmbH, Braunschweig, Germany). Sections were dewaxed and rehydrated using tap water RT. Antigen retrieval was performed using 0.1% Trypsin (Sigma-Aldrich, St. Louis, MO, USA) dissolved in preheated Tris-buffered saline (TBS) containing 0.1 % CaCl 2 (Merck, Darmstadt, Germany) for 45 min at 37 ◦ C. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide (Sigma-Aldrich, St. Louis, MO, USA) in methanol (VWR, Monroeville, PA, USA), 10 min at RT. Slides were washed with tap water at RT, and then non-specific binding was blocked using 10% normal goat serum (Vector Laboratories) containing 3% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA) for 15 min at RT. For the first stain, tissue sections were incubated with rabbit polyclonal antibody anti-Iba1 (FUJIFILM Wako, Osaka, Japan) for specific macrophage identification (Iba1 is largely restricted to cells of monocyte/macrophage lineage [27] at a 1:1200 dilution overnight at 4 °C and then washed three times with TBS 1X (1X TBS, 5 mM Tris (Merck KGaA, Darmstadt, Germany)/HCl (Panreac Química, SLU, Barcelona, Spain) pH 7.6, 136 mM NaCl (Merck KGaA, Darmstadt, Germany)) at RT. After that, sections were incubated for 30 min at RT with a biotinylated anti-rabbit IgG secondary antibody produced in goat (Vector Laboratories, Burlingame, CA, USA) at 1:200 dilution and slides washed as previously described. For signal detection sections were incubated for 30 min at RT with Avidin-Biotin Complex (ABC kit Peroxidase (PO) Standard, Vector Laboratories) followed by three washes with 1X TBS and incubation with 3,3`-Diaminobenzidine tetrahydrochloride (DAB) (Sigmafast, Sigma-Aldrich, St. Louis, MO, USA) for 2 min at RT. Afterwards, samples were rinsed with tap water for 5 min and washed with 1X TBS three times at RT. It is important to carry out the double-staining procedure in the specified order, using as first substrate the DAB. This sequence was previously recommended by other authors, because the DAB reaction product, a widely recognized chromogen, effectively masks and prevents unspecific cross-reactions [28]. Then, for the second D-IHC stain, the same slides were incubated overnight at 4 °C with rabbit anti-cytokeratin polyclonal (Dako, California, USA) at a 1:1000 dilution. It is a wide-spectrum screening antibody that detects low-molecular-weight CKs (40-54 kDa), specifically KRT7-8 and KRT17-20 according to Moll’s designation, and high-molecular-weight CKs (48-67 kDa), specifically KRT1-6 and KRT9-16. Samples were washed three times with 1X TBS at RT. Bounded antibody was detected by incubation for 30 min at RT with alkaline phosphatase (AP)-conjugated goat anti-rabbit IgG secondary antibody (Sigma-Aldrich, St. Louis, MO, USA) followed by three washes with 1X TBS and 1-StepTM NBT/BCIP (Thermo Scientific, Rockford, USA) for 6 min at RT under microscopic control. There was no need to block endogenous AP activity. Samples were rinsed with tap water for 5 min at RT and counterstained in Mayer’s hematoxylin (MerckKGaA, Darmstadt, Germany) for 5 seconds before washing, dehydrating, and mounting with DPX (Merck KGaA, Darmstadt, Germany). Single-Iba1 positive cells stained brown (DAB), single-CK positive cells stained blue (1-StepTM NBT/BCIP), and double Iba1/CK-positive cells stained dark blue or black. Because the AP substrate (1-StepTM NBT/BCIP) and Avidin-Biotin PO substrate (DAB) can produce a widespread precipitate over the entire section, both reagents were freshly prepared and micropore filtered 0.22 µm (Merck Millipore, Cork, Ireland) immediately before application to the tissue. Prior to the D-IHC optimization, a single immunohistochemistry (S-IHC) experiment was carried out to evaluate positive CK staining in the experimental samples (DJE and JELN) using as a CK-positive epithelial control tissue bovine endometrium [29,30] (Table 1). In bovine endometrium, a positive control (Control 1) without omission of any reactive was carried out, and a negative control (Control 2) performed with omission of primary antibody. Moreover, negative controls (Control 3) performed with omission of anti-CK primary antibody were analysed in experimental samples from animals with different types of PTB-associated lesions. Two negative controls and one positive were included in the D-IHC experiment (see Table 1). The first negative control (Control 4) consisted of slides processed as previously described for D-IHC with omission of the two primary antibodies used to detect unspecific binding of the secondary antibodies to the tissue, the second negative control (Control 5) consisted of slides treated as previously described with omission of the anti-CK primary antibody to detect cross-reaction between the two staining steps, and the positive control (Control 6) was performed without omission of any primary or secondary antibody. Image acquisition and quantification Immunolabelled sections from D-IHC were observed using an Olympus BH-2 light microscope (Olympus, Tokyo, Japan). Individual images were acquired using an Olympus DP-12 digital camera (Olympus, Tokyo, Japan). Images were taken throughout histological sections selecting fields with granulomatous lesions avoiding areas containing preparation artefacts, cell debris, or the edges of the slide. In control animals, although granulomas are not present, counting was carried out in equivalent regions of the preparations, in other words, the images were taken in areas where granuloma formation usually occurs, such as the cortical and paracortical area in the JELN and the apical area of the villi in the DJE or more precisely on villous tips. To evaluate cytokeratin expression in cattle with different histopathological forms of PTB, the number of single and double positive stained cells within granulomatous lesions and controls in each tissue sample from each individual was counted in ten randomly selected fields per individual at a final magnification of 400X so a total of 50-70 fields were examined for each histopathological type. Each field corresponded to one image captured using a 40X objective (1600 x 1200 pixels) having a theoretical area of 41207.52 µm2 (234,4 µm length x 175,8 µm height). The number of positive immunolabelled cells was manually counted in each selected field/image using the ImageJ program (National Institutes of Health, EEUU). Results are expressed as a mean of the number of positive cells per field ± standard deviation (SD) for each histopathological group. Positive cells have been classified into single-CK positive cells, single-Iba1 positive cells and double-Iba1/CK positive cells. Statistical Analysis Statistical analysis was carried out using the R program (R Development Core Team, version 4.1.3). Differences of quantitative variables between two groups was carried out using the Student´s t-test or Wilcoxon test for independent samples, depending on normality hypothesis. Differences between three or more groups were analysed using the ANOVA or Kruskal-Wallis´s test, depending on whether the hypotheses of homoscedasticity and normality were verified. When the Kruskal-Wallis´s test and the ANOVA´s tests were statically significant the non-parametric post hoc Dunn´s test and the parametric post hoc Tukey´s test, respectively, were conducted to determine exactly which pairwise groups had statically significant differences. The level of significance used was 0.05. Results Map infection status of studied animals The Map infection status of the 32 animals used in this study is shown in Table 2. The animals were classified into five groups according to the presence/absence and type of histological lesions in gut tissues (Gonzalez et al., 2005) (see Table 2): 1) the healthy control group (n=5), which consisted of animals with no observed PTB-associated lesions, negative by ELISA, and bacteriological culture and PCR of tissues and faeces; 2) the focal group (n=7) consisted of animals with focal lesions, all ZN positive and ELISA negative; 3) the multifocal group (n=7) included animals with PTB-associated multifocal lesions, positive by at least one of the diagnostic tests used. In this group, there was one animal, the only one showing clinical signs that was ZN+ but curiously it was negative by the rest of the tests; 4) the diffuse intermediate group (n=7) included animals with PTB-associated diffuse intermediate lesions with most animals positive by ELISA and PCR of faeces and tissues, and 71% of the animals showing clinical signs; and 5) the multibacillary diffuse group (n=6), which was composed of animals with multibacillary diffuse lesions with a large number of Map bacteria present, with 100% of the animals positive by ELISA and with PTB-associated clinical signs. Clinical signs were observed in 0%, 20 % and 85% of the animals with focal, multifocal and diffuse (intermediate and multibacillary) lesions (no information was available for 3 focal and 2 multifocal animals). No gross or histologic lesions compatible with other inflammatory processes were identified during post-mortem inspection of the studied animals. Assessment of the cross-reactivity and specificity of the immunoreagents used in the single and double-immunohistochemistry Positive CK staining was first evaluated by S-IHC in bovine endometrium used as a positive control for epithelial CK expression and in JELN and DJE of animals with different pathological forms of PTB (Table 1, Controls 1 to 3). In bovine endometrium, the positive control (Control 1), carried out without omission of any reactive, CK staining was observed exclusively in the cytoplasm of epithelial cells of the luminal epithelium, superficial glands within the stroma and Stratum basalis deep glands (Additional File 2A-C); the negative control (Control 2), performed with omission of the anti-CK primary antibody, no immunolabelled cells were observed (Additional File 2D-F). The anti-CK wide spectrum screening antibody used exhibited specificity in the positive bovine endometrium control and the biotinylated anti-rabbit IgG secondary antibody displayed no perceptible cross-reactivity with non-target proteins. In JELN and DJE samples CK staining was observed in all animals. In JELN (Additional File 3), positive cells were observed in the cortex and close to or forming part of granulomas (Additional File 3A-D), near connective tissue and trabeculae (Additional File 3E-H), and in the medullar area (Additional File 3I-L) for all type of PTB lesions. The number of CK-positive cells seemed to be more abundant toward the efferent lymphatic vessels (Additional File 3A, 3E, 3I for animals without lesions, 3B, 3F, 3J for focal animals, 3C, 3G, 3K for multifocal animals and 3D, 3H, 3L for intermediate diffuse animals). As the severity of PTB lesions increases so does the number of CK-positive cells, especially numerous in multifocal lesions (Additional File 3A-D in cortex, 3E-H in areas close to connective tissue and trabeculae, 3I-L in medullar area). In DJE (Additional File 4), CK-positive cells were detected in all animals scattered in the mucosa of DJE (Additional File 4A, 4E, 4I for animals without lesions, 4B, 4F, 4J for focal animals, 4C, 4G, 4K for multifocal animals and 4D, 4H, 4L for intermediate diffuse animals), throughout the lamina propria in the apical area (Additional File 4A-D and 4E-H), forming part of the granulomas (Additional File 4G and 4H), and around and in the crypts of Lieberkühn (Additional File 4I-L). As in the case of JELN samples, it appears that as the severity of the PTB-associated lesions increased, CK-positive cells became more numerous, especially in multifocal lesions (Additional File 4A-D in apical area, 4E-H in apical granulomas or equivalent area and 4I-L in basal area of DJE). No staining was observed when the anti-CK antibody was omitted (Control 3 of Table 1, results not shown). Cross-reactivity and specificity of detection antibodies in the D-IHC of JELN and DJE tissue samples was assessed analysing three controls (Table 1, D-IHC controls 4 to 6). In this assay, we expected to observe three types of positive immunolabeled cells (single-CK (light blue), single-Iba1 (brown) and double-Iba1/CK (dark blue) positive cells) and negative immunolabelled cells. In the negative control (Control 4) performed with omission of the two primary antibodies (anti-Iba1 and anti-CK) no positive cells were observed (Additional Files 5A and 5D for JELN and 6A and 6D for DJE) indicating that secondary antibodies displayed imperceptible cross-reactivity with non-target proteins and that PO and AP substrates did not show unspecific reactivity. In the negative control carried out with omission of the anti-CK primary antibody (Control 5) no single-CK (light blue) or double-Iba1/CK positive cells (dark blue) were observed (Additional Files 5B and 5E for JELN and 6B and 6E for DJE) indicating that in the sequential D-IHC procedure design the goat anti-rabbit IgG AP-conjugated secondary antibody did not bind unspecifically to anti-Iba1 primary antibodies; no cross-reactivity was detected between the two staining sequences. In the D-IHC positive control (Control 6), carried out without omission of any reactive, the three types of positive cells were observed within the granuloma (Additional Files 5C and 5F for JELN and Additional Files 6C and 6F for DJE). In the DJE positive control, some single-CK light blue immunostaining was observed out of the granulomas in the villi as part of the normal expression of CK in the intestine. These results demonstrate that the procedure of the D-IHC worked well in both tissue types, with no cross-reactivity observed between detection antibodies. Morphological analysis, distribution, and patterns of Iba1 and cytokeratin expressing cells in jejunal lymph nodes and distal jejunum of cattle with different histopathological forms of bovine paratuberculosis. The enrichment of the keratinization pathway in cattle with PTB-associated multifocal lesions was investigated at the protein level within granulomas of JELN and DJE samples, by double-Iba1/CK immunohistochemistry and quantification of the number of CK-positive EMs in animals with different pathological forms of PTB (n=25) and control animals without lesions (n=5). D-IHC analysis showed four types of cells within the granuloma and around the structure of both JELN and DJE samples (Figures 1 and 2, respectively): single-Iba1 positive macrophages stained as brown cells (Figure 1A for JELN and 2A for DJE), single-CK positive cells detected as bright blue cells (Figure 1B for JELN and 2B for DJE), double-Iba1/CK positive macrophages expressing CK observed as dark blue or black cells (Figure 1C for JELN and 2C for DJE), and negative cells (no immunolabelled cells). In JELNs (Figure 1 and 3), both expected CK-positive cell types (single-CK and double-Iba1/CK positive cells) were observed in all the samples (N=32), including the control samples, with no morphological differences observed in these cell types between animals of different groups and ages. Single-CK immunolabelled cells (bright blue) in the JELN were observed in a highly variable number as part of the granuloma and scattered in the cortex (Figure 1D) and sometimes near blood vessels, supporting tissue and trabeculae (TB) (Figure 1E) and medullar area (Figure 1F). Single-CK positive cells had medium to large-sized nuclei (4.22-8.65 µm in diameter) with a semi-round shape and sparse cytoplasm compatible with reticular cells (RCs) (similar morphology and localization) (Figure 1B). Double-Iba1/CK positive cells had large round to oval nuclei (4.36-9.12 µm in diameter) and abundant cytoplasm (Figure 1C). These double-immunolabeled cells were in the cortex of the lymph nodes in a scattered manner (Figure 1D), around the lymphoid follicles, as well as close to the germinal centre and in the subcapsular sinus. These cells were also observed in the paracortex area and especially in the medullary area tending to be more numerous toward the efferent lymphatic vessels in both infected and uninfected animals (Additional File 5C and 5F). Control animals without lesions showed a similar pattern of CK-expression to that mentioned above (Figure 3A and 3B), however as the severity of the PTB-associated lesions increases, the number of double-Iba1/CK positive cells increases in the cortex area, showing two different patterns, either double-Iba1/CK positive cells were found surrounding the granuloma or they were within the granuloma forming the granuloma itself (Additional File 7). The first scenario was more common in animals with focal and multifocal lesions (Figure 3C, 3D, 3E and 3F) while the second one seemed to be more frequent in animals with diffuse intermediate and diffuse multibacillary lesions (Figure 3G, 3H, 3I and 3J). These two patterns could be also observed in single CK-immunochemistry (See Additional File 3) confirming the results of the D-IHC. Multinucleated giant cells, both CK-positive (Figure 3G and 3I) and CK-negative, were also observed in JELN. With respect to single-Iba1 positive macrophages (stained as brown cells), these cells present large round to oval nuclei (5.11-9.83 µm in diameter) and abundant cytoplasm (Figure 1A) with a similar appearance to tissue macrophages that had the expected distribution. In DJE (Figure 2 and 4), single-Iba1, single-CK and double-Iba1/CK positive cells were also observed in all animals. Single CK-stained cells had medium- to large -sized round nuclei with scarce cytoplasm (Figure 2B). These cells were observed in a highly variable number as part of the granulomas and scattered in the mucosa (Figure 2D-E) and submucosa (Figure 2F) of DJE Double-Iba1/CK positive, with large round to oval nuclei and abundant cytoplasm (Figure 2C), were located scattered throughout the lamina propria mostly in the apical area (Figure 2D), around and in the crypts of Lieberkühn (Figure 2E), surrounding the villi (Figure 4B, 4C and 4D). As in the case of JELN samples, control animals without lesions did not show a striking pattern of CK expression (Figure 4A and 4B), but it appeared that as the severity of the PTB-associated lesion increased so did the number of double-Iba1/CK positive cells in the granuloma area, the granulomas showing the same two patterns previously described (Figure 4F where double-Iba1/CK positive cells are found surrounding the granuloma and 4H where they were within the granuloma). These two patterns could be also observed in single CK-immunochemistry (See Additional File 4G and 4H). Evaluation of cytokeratin expression at the protein level in granulomatous lesions of cattle with different histopathological forms of bovine paratuberculosis. The results of the quantification of the number of single and total-Iba1, single and total-CK and double-Iba1/CK positive cells in JELN are shown in Table 3. With respect to the number of single-CK positive cells (cells expressing cytokeratin that are not apparently macrophages) per field and histological group, significant differences were observed between the different histopathological groups (Kruskal-Wallis test, p <0.001). Specifically, post hoc Dunn´s test reported differences between the medians of the multifocal group (5.00 (2.00-14.00)) with the focal (3.50 (0.25-5.55)) and the control (2.00 (0.00-6.00)) groups (p=0.015 and 0.002, respectively). Regarding the total number of macrophages (single-Iba1 cells) no significant differences were observed between groups (Krustal-Wallis, p-value=0.504). As for the mean number of double-immunolabelled cells (Iba1 cells expressing CK, EMs) per field the group of animals with multifocal lesions showed the highest mean number (51.59 ± 24.40) and the control group the lowest (29.64 ± 20.52). In this case, significant differences between the cows with multifocal lesions and the cows with focal, diffuse intermediate, diffuse lesions (including intermediate and multibacillary) and controls (Tukey´s test, p<0.01, p<0.01, p=0.005 and p<0.01, respectively). The multifocal group had higher numbers of double-immunolabeled cells than the diffuse multibacillary group, however, no significant differences were observed (p=0.165). As for the total number of CK-expressing cells (single and double) and in comparison, with the number of single-CK-expressing cells, the main difference is that when we analysed the total number significant differences of the multifocal with the diffuse group (p=0.023) were now observed. Overall, the significance level of the differences observed between groups was higher when we compared numbers of double-Iba1/CK immunolabelled cells. No significant differences were observed in the total number (single and double) of Iba1 positive cells between groups (ANOVA, p-value=0.185). Significant differences in the mean values of single-CK, double-Iba1/CK and total CK- expressing cells were also observed between infected animals with any type of lesions (focal, multifocal and diffuse animals) and control animals without lesions (Welch´s test p<0.001, Student´s test p=0.001 and Welch´s test p<0.001, respectively), showing the control animals lower mean numbers than the infected animals with any type of lesion. The levels of cytokeratin expression in granulomas of cattle with different histopathological forms of bovine paratuberculosis was also evaluated in DJE samples (Table 4). Regarding single-CK positive cells, significant differences were found between infected animals (focal, multifocal and diffuse) and control animals (Test of Welch, p-value=0.045) and between the multifocal and the focal groups (Dunn´s test, p-value=0.002), showing the multifocal group the highest numbers of immunolabelled cells. No significant differences between pairs of histopathological groups were observed for single-Iba1 positive cells, double-immunolabeled cells and total number of CK-immunolabeled cells. Significant differences were detected in the total number of Iba1-positive cells between the multifocal and the diffuse intermediate groups. In order to shed light on the consequences of epithelioid transformation of macrophages in Map infection and disease progression the ratios of EMs (double Iba1/CK positive cells) with respect to non-epithelioid macrophages (non-EMs) (single-Iba1 positive cells) and the total number of macrophages (single and double-Iba1 cells) was also investigated (Table 5). In JELN it was found that the ratios of EMs/non-EMs were lower than one (0.39-0.75) indicating that the number of non-EMs (single Iba1-positive cells) was higher than the EMs, with multifocal animals showing the highest ratio of EMs/non-EMs (higher numbers of EMs than the rest of the histopathological groups). Likewise, infected animals showed higher ratios than control animals. In DJE, all the histopathological groups present similar ratios of EMs/non-EMs, which were close to 1 indicating that they had similar amounts of EMs and non-EMs. In DJE lower total numbers of non-EMs and higher of EMs than in JELN were observed. Discussion The knowledge of the composition and structure of the granuloma, made up of macrophages in different stages of differentiation and other immune cells, may help to understand the role of the granuloma in the control of Map infection. In the present study, the number and distribution pattern of CK-expressing cells in the granulomas of animals with different pathological forms of bovine PTB was analyzed for the first time to investigate the role of epithelioid granulomas in the control of Map infection. Our results show that CK expression was enriched in granulomas of cattle with multifocal lesions. Multifocal animals had significantly higher numbers of double CK/Iba1 positive EMs in JELN granulomas (Table 3 ) and higher numbers of single-CK expressing cells in JELN and DJE granulomas (Tables 2 and 3 ). Likewise, infected animals had significantly higher levels of single and double-CK positive cells than control animals. In DJE, no significant differences in the number of EMs were observed between histopathological groups or between infected and non-infected control animals. However, significant differences in the number of single-CK cells were observed between infected and non-infected animals. The number of macrophages in the control group without lesions it is quite high in comparison with the rest of the evaluated histopathological groups. This is expected as the gastrointestinal system has the largest number of tissue macrophages in the intestine [ 31 ]. The high number of EMs could be explained because in natural infections animals may be exposed to other immunological stimuli, which may affect epithelioid transformation of macrophages [ 32 ]. Regarding EMs, two different distribution patterns were observed (Additional File 7) in JELN and DJE. Granulomas of multifocal animals presented an ordered structure where EMs were forming a barrier surrounding the granuloma. Moreover, multifocal animals had significantly lower infection scores than diffuse animals and most animals were subclinical. These findings suggest that this barrier is preventing dissemination of Map and maintaining tissue integrity, playing a relevant role in controlling infection. Animals with diffuse lesions, where CK-expressing EMs were observed throughout the granuloma and did not form a barrier around them, had higher titers of Map, showed clinical signs and were not able to contain the infection efficiently despite having high titers of anti-Map antibodies. Therefore, the number and especially the distribution pattern of EMs in the different histopathological groups had an effect of in the control of Map and in the development of clinical disease. Various hypotheses about the consequences of epithelioid transformation of macrophages have been postulated, some authors believe that it engenders highly phagocytic and microbicidal [ 33 ] while others suggest that it produces non-phagocytic cells with secretory functions that enhance the immune response [ 34 , 35 ]. A longitudinal study [ 36 ] of tuberculous granulomas induced by chronic M. marinum infection of frogs revealed that both types of epithelioid cells (phagocytic and microbicidal cells/ non-phagocytic cells with secretory functions) could be present in chronic granulomas, showing that, even long-term granulomas (long subclinical stage) are dynamic environments where bacterial replication and their phagocytic clearance maintain relatively stable bacterial numbers despite the established immune response. Even though the granuloma tends to become more epithelioid with time, even in the most chronic phases, infected and highly activated non-EMs were found together with epithelioid cells. In the present study, non-EMs and EMs were found in DJE and JELN showing similar numbers in DJE while higher numbers of non-EMs were observed in JELN. Further experiments should be performed to investigate the presence of Map in macrophages and the types and function of EMs (phagocytic and microbicidal cells/ non-phagocytic cells with secretory functions) in the granulomas of Map-infected animals. Tissue-resident macrophages are extremely heterogeneous in a manner that is suited to tissue-specific [ 37 ]. The differences observed between DJE and JELN could be due to the fact that DJE and JELN have different functions while one of the fundamental functions of DJE is tissue homeostasis involving macrophages that participate in tissue repair and resolution of inflammation the main function of JELN is immunosurveillance, the heterogeneity of macrophages due to these functions may result in changes in epithelioid differentiation processes. Moreover, gut macrophages in DJE have a half-life estimated of 3 weeks [ 38 ], which means that cells are in a continuous renewal, affecting the accumulation of epithelioid macrophages which might not allowed to detect differences between groups. The role of the granuloma in mycobacterial infections, such as tuberculosis, has been previously revised [ 39 ]. Historically, it has been regarded as a host-protective structure that walls off the infecting mycobacteria, constituting a barrier to bacterial proliferation and dissemination. In contrast, an opposing trend has emerged that presumes that the granuloma is a highly dynamic structure promoting bacterial proliferation [ 40 – 42 ]. I has been shown that mycobacterial granuloma formation in a Zebrafish- Mycobacterium marinum model is accompanied by macrophage induction of canonical epithelial molecules and structures [ 43 ]. They demonstrated that disruption of macrophage epithelial protein biomarkers such as E-cadherin, resulted in disordered granuloma formation which facilitates and enhances immune access, resulting in a decrease of bacterial burden and increase host survival suggesting that the granuloma has a bacteria-protective role. They suggested that E-cadherin expressing macrophages could be acting as an access barrier to protect bacteria from the host immune system in a similar structure disposition to the one observed in the present study in animals with focal and multifocal histological lesions where EMs were found surrounding the granuloma. However, in our study (Table 2 ), animals presenting this EMs disposition in the granuloma were subclinical and had low titers of Map indicating that the barrier seems to be controlling infection rather than producing a favorable niche for mycobacteria. In leprosy, it has been shown that the development of a robust immune response results in paucibacillary disease, where well-developed granulomas contain scant organisms, whereas in multibacillary leprosy with poor to no granuloma formation high bacterial growth is observed. However, paucibacillary leprosy sometimes causes severe morbidity, indicating that an overexuberant granulomatous response is not good either, while restricting bacteria also harms the host [ 44 ]. Curiously, animals with multibacillary leprosy, not able to control mycobacteria replication, present down-regulation of the expression of keratinocyte-associated genes (KRT14 and KRT5) [ 45 ]. In tuberculoid leprosy, the presence of epithelioid giant cells and the formation of granulomas leads to the control of Mycobacterium leprae replication and the containment of its spread, however, in lepromatous lesions phagocytic foamy macrophages are observed and are unable to control M. leprae replication [ 46 ]. The presence of non-EMs (Iba1-positive cells) was also investigated in DJE and JELN granulomas of the different histopathological groups and no significant differences were observed between histopathological groups and between non-infected and control animals in the number of Iba1-positive cells. In contrast, Jenvey et al. (2019) [ 47 ] showed that the number of macrophages in the mid ileum increases with progression of infection even though a significant number of macrophages were not associated with Map. This finding suggests that although the infected animal recruits more macrophages to the site of infection, these increased numbers of macrophages are unable to clear Map, which is transmitted from macrophage to macrophage leading to further progression of the infection, and the development of clinical disease. Even though our results are different from those reported by Jenvey et al. (2019) [ 47 ], it must be noted that we have used samples from a different section of the gut tissue (frozen versus (vs) fresh, mid ileal vs . DJE and JELN) as well as a different detection technique (immunohistochemistry vs immunofluorescence), primary antibody (anti-Iba1 vs anti-macrophage surface antigen (clone AM-3- K)) and quantification protocol (mean labelled area in mid ileal tissue section vs mean number of macrophages in granulomas per field; in our case counting is restricted to granulomas in contrast with counting the total tissue section as in Jenvey et al.) [ 47 ]. It is possible that we are also counting other cells of the monocyte/macrophage lineage using anti-Iba1 antibody [ 27 ]. During maturation several additional cell types are recruited within the granuloma that can define the impact and function of the granuloma within the infection or disease. In this sense, we have observed the presence of single-CK positive cells in the granulomas that showed similar morphology and localization to RCs, which are easily identified using cytokeratin as a marker. Here, higher numbers of single-CK expressing cells were observed in JELN and DJE granulomas of multifocal animals. Previous studies in humans showed the presence and distribution of CK-immunoreactive reticular cells in normal and pathological human lymph node [ 48 , 49 ]. RCs are normally present in lymphoid organs such as lymph nodes and play a crucial role in immune response to infection or inflammation [ 50 ]. Moreover, RCs can constrain an excessive immune response [ 51 ]. RCs were shown to be involved in transplantation tolerance, cancer immunity and they are considered as a key factor in autoimmune diseases [ 52 , 53 ], which have been associated with Map infection. In this sense, RCs could be also helping to control Map infection, however, this is beyond the scope of the current study. Continuous exposure of animals to Map results in a dynamic balance where infection never gets established or is controlled by an efficient innate immune response in an important part of the farm population while in other individuals infection progresses to subclinical delimited focal or multifocal forms and in a smaller fraction of the herd to diffuse lymphocytic (cellular or Th1 type) or non-lymphocytic (humoral immune response or Th2 type) forms that will result in clinical disease [ 54 ]. Cellular immunity may exert some control over the infection at early stages, but this appears to be very different between individual cows, with different consequences on shedding and the development of a humoral response over [ 55 , 56 ]. It might be possible that one of the factors involved in this switch from a Th1 to a Th2 response is the activation stage and organization of macrophages in the granulomatous lesion itself. It is possible that an increase in the number of EMs located around the granuloma forming an epithelioid barrier might inhibit granuloma growth by disrupting the recruitment of newly activated macrophages to the structure hindering mycobacterial macrophage-to-macrophage spread and thus serving to reduce their intracellular niche, favouring equilibrium towards EMs and walling off the bacteria inside the granuloma. In summary, the number and distribution pattern/organization of EMs in the granuloma have a relevant role in controlling Map infection. Multifocal animals can control infection through formation of granulomas where EMs expressing CK, localized around the granuloma, form a barrier that limits/repairs tissue damage protecting animals from clinical disease. Our results agree with the findings of Canive et al. (2021) [ 21 ] where a significant enrichment of the KRT pathway was observed, at the gene expression level, in animals with multifocal lesions suggesting that KRT genetic variants may predispose cattle to develop multifocal lesions that maintain tissue resilience through rearrangements in the keratin filaments. It might be considered that animals that form this type of granuloma, might be resilient to the disease, in terms of controlling the shift from subclinical to the clinical disease. Whether this control is long-lasting needs to be further investigated, since there might exist external factors that trigger the loss of this control at the granuloma level, and therefore the progression of the disease. Our findings suggest that cytokeratin could be considered as a potential biomarker of PTB resilience. Tolerance is defined as the mechanisms used by the host to reduce susceptibility to tissue damage and other fitness damages caused by pathogens or immune response, so, in this sense, cytokeratin could be considered specifically as a biomarker of tolerance. Declarations Ethics approval and consent to participate Animals used in this study had their origin in commercial farms. All farmers were informed about the study and gave their consent and approval for the use of samples in the present study. This study was carried out in accordance with Directive 2012/63/EU of the European Parliament. Experimental procedures were evaluated by the SERIDA Animal Ethics Committee board approval and authorized by the Regional Consejería de Agroganadería y Recursos Autóctonos del Principado de Asturias, Spain (authorization codes PROAE 29/2015 and PROAE 66/2019). Consent for publication Not applicable. Availability of data and materials All data generated or analysed during this study will be available from the corresponding author on reasonable request. Competing interests None of the authors of this manuscript have any competing or financial interests. Funding This study is part of the I+D+I project (PID2021-122195OR-C22), which was funded by the MCIN/AEI /10.13039/501100011033 / FEDER, UE and by regional funds PCTI 2021–2023 (GRUPIN: IDI2021-000102). We acknowledge the National Institute for Agricultural Research (INIA) for the scholarships of Cristina Blanco Vázquez (CPD2016.0142 funded by MCIN/AEI/10.13039/50110001033 and FSE) and Alejandra Isabel Navarro León (PRE20200-096451). The funding bodies have not been involved in the design of the study and collection, anlaysis, and interpretation of data and in writing the manuscript. Author´s contributions Design of the study: RC and AINL. Perform experiments: AINV, MM, CBV, NI, and TI. Wrote the manuscript: RC and AINL. 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BSA, bovine serum albumin; TBS; 1X TBS (5 mM Tris/HCl pH 5.6, 136 mM NaCl); AP, alkaline phosphatase; DPX, dibutylphthalate polystyrene xylene. Additionalfile2.tif Additional File 2. Representative images of Cytokeratin (CK) single-immunohistochemical (S-IHC) labelling in bovine endometrium of a Holstein Friesian cow used as CK-positive control. A to C display positive controls carried out without omission of any reactive: A) show positive labelling in the luminal epithelium and superficial glands within the stroma B) offers a closer view 200X magnification of superficial endometrial glands. The bar represents 50 microns; C) displays positive labelling in the stratum basalis deep glands of bovine endometrium; D to F show negative control performed with omission of primary antibody: D) displays negative labelling in the luminal area of endometrium. E) presents a magnified view (200X) of endometrial glands. The bars represent 50 microns; and F) shows negative labelling in the basal area of the endometrium. All sections, excluding B) and E) were examined at 100X magnification and the bars represent 100 microns. CK immunolocalization was investigated using a broad-spectrum rabbit polyclonal anti-CK primary antibody (Dako, California, USA). In this particular assay 3,3- Diaminobenzidine (DAB) was used as chromogen for CK-Staining (brown cells). Additionalfile3.tif Additional File 3. Detection of cytokeratin (CK) pattern expression in jejunal lymph nodes by single-immunohistochemistry (S-IHC) of animals with different histopathological forms of PTB and control animals without lesions. Three areas of interest are depicted in the images: A to D show cortex areas of the JELN at 200X magnification where presence of granulomas is frequent: A) control cow with no detected lesions; B), C), and D) animals with focal, multifocal, and diffuse intermediate lesions in their intestinal tissues, respectively.E to H display areas of JELN where connective tissue or trabeculae are present at 200X magnification: E) control cow; F), G), and H) show animals with focal, multifocal, and diffuse intermediate lesions, respectively. I to L show images of medullar areas of the JELN at 200X magnification: I) control cow with no lesions detected; J), K), and L) animals with focal, multifocal, and diffuse intermediate lesions. The bars represent 50 microns. CK immunolocalization was carried out using a broad-spectrum rabbit polyclonal anti-CK antibody (Dako, California, USA). TB, trabeculae; CT, connective tissue; C, capsule. Yellow circles show the distribution pattern of the CK-positive cells in the granuloma. Additionalfile4.tif Additional File 4. Detection of cytokeratin (CK) pattern expression in distal jejunum by single-immunohistochemistry (S-IHC) of cows with different histopathological forms of PTB and control animals without lesions. Three areas of interest are depicted in the images. A to D show villi apical areas of the DJE at 200X magnification where formation of granulomas is frequent: A) control cow with no lesions detected; B), C), and D) animals with focal, multifocal, and diffuse intermediate lesions in their intestinal tissues, respectively. E to Hdisplay at a higher magnification (400X) the apical area likely to be affected by granulomas: E) control cow without granulomas; F) focal cow with no granulomas observed; G), and (H) animals with granulomas of multifocal, and diffuse type, respectively. I-L show basal areas of the lamina propia of DJE at 200X magnification: I) control cow with no lesions detected; J), K), and L) show animals with focal, multifocal, and diffuse intermediate lesions.The bars represent 20 microns in images E-H and 50 microns in the rest of the images. CK immunolocalization was carried out using a broad-spectrum rabbit polyclonal anti-CK antibody (Dako, California, USA). Yellow circles show the distribution pattern of the CK-positive cells in the granuloma. additionalfile5.tif Additional File 5. Detection of cytokeratin (CK) and Iba1 expression in jejunal lymph nodes (JELN) of one of the cows included in the diffuse multibacillary group by double-immunohistochemistry (D-IHC). A-C show roughly equivalent areas of the JELN cortex where formation of granulomas is frequent: A) D-IHC control 4 performed with omission of both primary’ antibodies; B) D-IHC control 5 performed with omission of the anti-CK primary antibody to detect cross-reaction between the two staining sequences; C) D-IHC control 6 without omission of any reactive. D-F display roughly equivalent areas of the JELN medulla: D) D-IHC control 4 performed with omission of both primary’ antibodies; E) D-IHC control 5 performed with omission of the anti-cytokeratin primary antibody to detect cross-reaction between the two staining sequences; F) D-IHC control 6 without omission of any reactives. The standard D-IHC demonstrate the correct functioning of the protocol used to carry out the D-IHC. Double- Iba1/CK cells (dark blue) as well as some single-Iba1 (brown cells) and single-CK (bright blue) (Images C and F) can be observed. Omission of anti-CK primary antibody reveals absence of non-specific binding for the second staining sequence of the protocol (images B and E). Omission of both primary antibodies demonstrates absence of non-specific binding for the whole procedure (images A and D). The bars represent 50 microns and sections were examined at 200X magnification. The C in red points to the capsule. For controls 4, 5 and 6 (Table 1). Additionalfile6.tif Additional File 6. Detection of cytokeratin (CK) and Iba1 expression in distal jejunum (DJE) of one cow with diffuse multibacillary lesions by double-immunohistochemistry (D-IHC). A-C show roughly equivalent villi apical areas of the DJE where formation of granulomas is more frequent: A) D-IHC control 4 performed with omission of both primary’ antibodies; B) D-IHC control 5 performed with omission of the anti-cytokeratin primary antibody to detect cross-reaction between the two staining sequences; and C) D-IHC control 6 without omission of any reactives. D-F display equivalent basal areas of the lamina propia of the DJE: D) D-IHC control 4 performed with omission of both primary’ antibodies; E) D-IHC control 5 performed with omission of the anti-CK primary antibody to detect cross-reaction between the two staining sequences; F) standard procedure of D-IHC control 6 without omission of any reactives. Strong double- Iba1/CK cells (dark blue) as well as some single-Iba1 (brown cells) and single- CK (bright blue) (Images C and F) can be observed; some single-CK light blue immunostaining out of the granulomas that may seem unspecific is observed in the villi but this staining is part of the normal and known expression of CK in the intestine. Omission of anti-cytokeratin primary antibody reveals absence of non-specific binding for the second staining sequence of the protocol (images B and E). Omission of both primary antibodies demonstrates the absence of non-specific binding for the whole procedure. The bars represent 50 microns and sections were examined at 200X magnification. For controls 4, 5 and 6 (Table 1). Additioanalfile7.tif Additional File 7. Schematic representation of the structure and cellular components of the two distribution patterns of double Iba1/CK- positive cells (EMs) observed in the granuloma of Map infected animals with different pathological forms of PTB. A) Granuloma of multifocal animals and B) Granuloma of diffuse animals. Schemes generated by BioRender.com (2020). Retrieved from href="https://app.biorender.com/biorender-templates">https://app.biorender.com/biorender-templates Tables.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3982091","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":298901957,"identity":"4f781355-fb90-4187-8c2e-e5d6efcba14e","order_by":0,"name":"Alejandra Isabel Navarro León","email":"","orcid":"","institution":"SERIDA: Servicio Regional de Investigacion y Desarrollo Agroalimentario","correspondingAuthor":false,"prefix":"","firstName":"Alejandra","middleName":"Isabel Navarro","lastName":"León","suffix":""},{"id":298901958,"identity":"74853b05-3364-49aa-9e50-451176a7b3f7","order_by":1,"name":"Marta Muñoz","email":"","orcid":"","institution":"SERIDA: Servicio Regional de Investigacion y Desarrollo Agroalimentario","correspondingAuthor":false,"prefix":"","firstName":"Marta","middleName":"","lastName":"Muñoz","suffix":""},{"id":298901959,"identity":"c633a70f-1ef3-459b-a65c-bd1121065ded","order_by":2,"name":"Cristina Blanco-Vázquez","email":"","orcid":"","institution":"SERIDA: Servicio Regional de Investigacion y Desarrollo Agroalimentario","correspondingAuthor":false,"prefix":"","firstName":"Cristina","middleName":"","lastName":"Blanco-Vázquez","suffix":""},{"id":298901960,"identity":"9606ce50-0204-4f3d-9ff3-ff0bd1786918","order_by":3,"name":"Natalia Iglesias","email":"","orcid":"","institution":"SERIDA: Servicio Regional de Investigacion y Desarrollo Agroalimentario","correspondingAuthor":false,"prefix":"","firstName":"Natalia","middleName":"","lastName":"Iglesias","suffix":""},{"id":298901961,"identity":"15f2b694-50ac-4e11-9fea-c2c991a79e13","order_by":4,"name":"Tania Iglesias","email":"","orcid":"","institution":"Universidad de Oviedo","correspondingAuthor":false,"prefix":"","firstName":"Tania","middleName":"","lastName":"Iglesias","suffix":""},{"id":298901962,"identity":"d46ea95c-a80b-4044-948d-f0d70156abbc","order_by":5,"name":"María Canive","email":"","orcid":"","institution":"NEIKER Basque Institute of Agricultural Research and Development: NEIKER Instituto Vasco de Investigacion y Desarrollo Agrario SA","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"","lastName":"Canive","suffix":""},{"id":298901963,"identity":"f3c53a41-ec4f-4643-a8a5-2f44e40a9978","order_by":6,"name":"Gerard Badia-Bringué","email":"","orcid":"","institution":"NEIKER Basque Institute of Agricultural Research and Development: NEIKER Instituto Vasco de Investigacion y Desarrollo Agrario 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Casais","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYPACGyBmbJAgRUsa6VoOg0nitJi3tz/88OPXeXt+6cONN34w2OTLOxDQInPmjLFkb9/txJl9ic2WPQxplhsPENAiIZHDIM3YczvB4AxjmwQPw2EDwwaCWtIf/2bsOWdvD9Qi+Yc4LQlm0gw/DjBu4GFskwbZIk9AB4MEzxkzy96G5MQZZxibrWUM0gwMCGphb39848cfO3v+HvaHN99U2BjIE3IYGDC2wVhAKwwOEKOF4Q8SmzhbRsEoGAWjYCQBACNFPMPr7cbwAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-8349-5878","institution":"Servicio Regional de Investigación y Desarrollo Agroalimentario: Servicio Regional de Investigacion y Desarrollo Agroalimentario","correspondingAuthor":true,"prefix":"","firstName":"Rosa","middleName":"","lastName":"Casais","suffix":""}],"badges":[],"createdAt":"2024-02-23 14:15:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3982091/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3982091/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56237403,"identity":"97809108-532c-4003-a408-19d0afb2f866","added_by":"auto","created_at":"2024-05-10 09:04:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":8632723,"visible":true,"origin":"","legend":"\u003cp\u003eDouble- ionized calcium-binding adapter molecule-1 (Iba1)/cytokeratin (CK) immunohistochemical analysis of jejunal lymph node (JELN) samples of a control cow without lesions showing the distribution and morphological appearance of the three immunolabelled cell types detected (CK-positive, Iba1-positive and double-Iba1/CK positive cells). A to C show the different immunolabeled cell types: A) single Iba1-positive cells (brown colour) in the cortex; B) single CK-positive cells (bright blue) in medulla; and C) double Iba1/CK positive cells (dark blue) in the cortex. D to F display different areas of distribution of the three cell types: D) cortex area of JELN close to the line of the subcapsular sinus; E) trabeculae at the high of the interfollicular cortex of JELN; F) medullar area of JELN. A-C, magnification 1000X, bars 10 µm. D-F, magnification 400 X, bars 20 µm. Red arrows point to single cells representing the three different immunolabeled cell types. C, capsule; TB, trabeculae.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3982091/v1/07df025b934b01c3be3007ef.png"},{"id":56237409,"identity":"541ea37e-b3c2-467d-a1d7-1825c605c599","added_by":"auto","created_at":"2024-05-10 09:04:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":8092885,"visible":true,"origin":"","legend":"\u003cp\u003eDouble- ionized calcium-binding adapter molecule-1 (Iba1)/cytokeratin (CK) immunohistochemical analysis of distal jejunum (DJE) samples of a control cow without lesions showing the distribution and morphological appearance of the three immunolabelled cell types detected (CK-positive, Iba1-positive and double-Iba1/CK positive cells). A to C show the different immunolabeled cell types: A) single Iba1-positive cells (brown cells) in the basal mucosa; B) single CK-positive cells (bright blue) in the apical mucosa; C) double Iba1/CK-positive cells (dark blue) in the apical mucosa. D to F display different areas of distribution of the three cell types in DJE: D) apical area of DJE mucosa; E) basal area of DJE mucosa; F) DJE submucosa. A-C, magnification 1000X, bars 10 µm. D-F, magnification 400X, bars 20 µm. Red arrows point to single cells representing the three different immunolabeled cell types.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3982091/v1/f56536cf0fb172e16fda5fcf.png"},{"id":56237407,"identity":"8d9560d1-1b67-4cec-a846-35d4e6fe926f","added_by":"auto","created_at":"2024-05-10 09:04:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":13652091,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative images of cytokeratin (CK) and Iba1 double-immunohistochemical (D-IHC) analysis of jejunal lymph nodes (JELN) samples of animals with different histopathological forms of PTB and control animals without lesions. (A, B) Control cow with no lesions detected; (C, D), (E, F), (G, H) and (I, J) show granulomas in animals with focal, multifocal, diffuse intermediate, and diffuse multibacillary lesions in their intestinal tissues, respectively. The bars represent 50 microns and sections were examined at 200X magnification. Yellow circles indicate resscorresponding to images B, D, F, H, and J. The bars represent 20 microns. CK and Iba1 immunolocalization was investigated using a broad-spectrum rabbit polyclonal anti-CK antibody (Dako, California, USA) and a rabbit polyclonal anti-Iba1 antibody (FUJIFILM Wako, Osaka, Japan). All granulomas depicted belong to the most JELN cortical zone. Red arrows point to examples of double-stained cells (dark blue or black); pink arrows point to single CK-positive cells (light blue); yellow arrows point to single Iba1-positive cells (brown colour); and green arrows point to multinucleated giant cells. Notice that CK-expression in animals with multifocal lesions show a pattern where epithelioid macrophages (EMs) are located around the granuloma (images E and F) while in animals with diffuse lesions a different pattern is observed where EMs are distributed through the whole granuloma (images G and H).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3982091/v1/98ca05db33edcf94fd3d6645.png"},{"id":56237404,"identity":"49b449a2-edf7-48f8-982b-b21a3ae29102","added_by":"auto","created_at":"2024-05-10 09:04:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":12900435,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative images of Cytokeratin (CK) and Iba1 double-immunohistochemical (D-IHC) results in distal jejunum (DJE) of infected cows with different histopathological forms of PTB and control cows without observed lesions. (A, B) Control cow with no detected lesions; (C, D), (E, F), (G, H) and (I, J) show granulomas in animals with focal, multifocal, diffuse intermediate, and diffuse multibacillary lesions in their intestinal tissues, respectively. The bars represent 50 microns and sections were examined at 200X magnification. Yellow circles indicate the areas that are shown in the right-hand column at a higher magnification (400X) corresponding to images B, D, F, H, and J. The bars represent 20 microns. All granulomas shown are located in the most DJE apical zone. Red arrows point to examples of double-stained cells (dark blue or black); yellow arrows point to single Iba1- positive cells (brown colour); pink arrows point to examples of single-CK stained cells (bright blue); and green arrows point to multinucleated giant cells. CK and Iba1 immunolocalization was investigated using a broad-spectrum rabbit polyclonal anti-CK antibody (Dako, California, USA) and a rabbit polyclonal anti-Iba1 antibody (FUJIFILM Wako, Osaka, Japan). Notice that CK-expression in animals with multifocal lesions shows a pattern where epithelioid macrophages (EMs) are located around the granuloma (images E and F) while in animals with diffuse lesions a different pattern is observed where EMs are distributed through the whole granuloma (images G to J).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3982091/v1/c1684883b41bfa20d4868c52.png"},{"id":61082873,"identity":"098a43d0-ce8d-4590-a038-5a9a2632b262","added_by":"auto","created_at":"2024-07-25 11:10:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":70551516,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3982091/v1/44223355-792a-4666-b509-eebcff802de0.pdf"},{"id":56237405,"identity":"0460e3a8-d945-491f-aac9-919cc3a40aa1","added_by":"auto","created_at":"2024-05-10 09:04:40","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1050578,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional File 1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScheme of the procedure followed to carry out sequential double (Iba1/CK)-immunohistochemical analysis of bovine jejunal lymph nodes and distal jejunum of cows with different pathological forms of bovine paratuberculosis and control animals without lesions.\u003c/strong\u003e RT, room temperature; CK, cytokeratin. BSA, bovine serum albumin; TBS; 1X TBS (5 mM Tris/HCl pH 5.6, 136 mM NaCl); AP, alkaline phosphatase; DPX, dibutylphthalate polystyrene xylene.\u003c/p\u003e","description":"","filename":"Additionalfile1.tif","url":"https://assets-eu.researchsquare.com/files/rs-3982091/v1/c9b3cba6306bb74bb120308e.tif"},{"id":56237410,"identity":"40a934ab-73ab-4392-85d0-efdcc1938eea","added_by":"auto","created_at":"2024-05-10 09:04:43","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12051322,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional File 2\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRepresentative images of Cytokeratin (CK) single-immunohistochemical (S-IHC) labelling in bovine endometrium of a Holstein Friesian cow used as CK-positive control\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eA to C display positive controls carried out without omission of any reactive: A) show positive labelling in the luminal epithelium and superficial glands within the stroma B) offers a closer view 200X magnification of superficial endometrial glands. The bar represents 50 microns; C) displays positive labelling in the \u003cem\u003estratum basalis\u003c/em\u003e deep glands of bovine endometrium; D to F show negative control performed with omission of primary antibody: D) displays negative labelling in the luminal area of endometrium. E) presents a magnified view (200X) of endometrial glands. The bars represent 50 microns; and F) shows negative labelling in the basal area of the endometrium. All sections, excluding B) and E) were examined at 100X magnification and the bars represent 100 microns. CK immunolocalization was investigated using a broad-spectrum rabbit polyclonal anti-CK primary antibody (Dako, California, USA). In this particular assay 3,3- Diaminobenzidine (DAB) was used as chromogen for CK-Staining (brown cells).\u003c/p\u003e","description":"","filename":"Additionalfile2.tif","url":"https://assets-eu.researchsquare.com/files/rs-3982091/v1/d949f7e6e2dff40d8234742e.tif"},{"id":56237415,"identity":"780a552e-aabe-42f0-8ff9-fcae6d11e842","added_by":"auto","created_at":"2024-05-10 09:04:46","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":19758742,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional File 3.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of cytokeratin (CK) pattern expression in jejunal lymph nodes by single-immunohistochemistry (S-IHC) of animals with different histopathological forms of PTB and control animals without lesions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree areas of interest are depicted in the images: A to D show cortex areas of the JELN at 200X magnification where presence of granulomas is frequent: A) control cow with no detected lesions; B), C), and D) animals with focal, multifocal, and diffuse intermediate lesions in their intestinal tissues, respectively.E to H display areas of JELN where connective tissue or trabeculae are present at 200X magnification: E) control cow; F), G), and H) show animals with focal, multifocal, and diffuse intermediate lesions, respectively. I to L show images of medullar areas of the JELN at 200X magnification: I) control cow with no lesions detected; J), K), and L) animals with focal, multifocal, and diffuse intermediate lesions. The bars represent 50 microns. CK immunolocalization was carried out using a broad-spectrum rabbit polyclonal anti-CK antibody (Dako, California, USA). TB, trabeculae; CT, connective tissue; C, capsule. Yellow circles show the distribution pattern of the CK-positive cells in the granuloma.\u003c/p\u003e","description":"","filename":"Additionalfile3.tif","url":"https://assets-eu.researchsquare.com/files/rs-3982091/v1/0cec4c8fe3d4d898a608169e.tif"},{"id":56237412,"identity":"66cd9de9-bd07-40dd-8643-a244266e46d9","added_by":"auto","created_at":"2024-05-10 09:04:43","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":16758112,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional File 4.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of cytokeratin (CK) pattern expression in distal jejunum by single-immunohistochemistry (S-IHC) of cows with different histopathological forms of PTB and control animals without lesions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree areas of interest are depicted in the images. A to D show villi apical areas of the DJE at 200X magnification\u003cstrong\u003e \u003c/strong\u003ewhere formation of granulomas is frequent: A) control cow with no lesions detected; B), C), and D) animals with focal, multifocal, and diffuse intermediate lesions in their intestinal tissues, respectively. E to Hdisplay at a higher magnification (400X) the apical area likely to be affected by granulomas: E) control cow without granulomas; F) focal cow with no granulomas observed; G), and (H) animals with granulomas of multifocal, and diffuse type, respectively. I-L show basal areas of the lamina propia of DJE at 200X magnification: I) control cow with no lesions detected; J), K), and L) show animals with focal, multifocal, and diffuse intermediate lesions.The bars represent 20 microns in images E-H and 50 microns in the rest of the images. CK immunolocalization was carried out using a broad-spectrum rabbit polyclonal anti-CK antibody (Dako, California, USA). Yellow circles show the distribution pattern of the CK-positive cells in the granuloma.\u003c/p\u003e","description":"","filename":"Additionalfile4.tif","url":"https://assets-eu.researchsquare.com/files/rs-3982091/v1/ef7036b7a764bb2a2d73eb76.tif"},{"id":56237413,"identity":"861895cd-120c-4e61-aa73-32c337809b65","added_by":"auto","created_at":"2024-05-10 09:04:45","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":16957142,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional File 5.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of cytokeratin (CK) and Iba1 expression in jejunal lymph nodes (JELN) of one of the cows included in the diffuse multibacillary group by double-immunohistochemistry (D-IHC).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA-C show roughly equivalent areas of the JELN \u003cstrong\u003ecortex\u003c/strong\u003e where formation of granulomas is frequent: A) D-IHC control 4 performed with omission of both primary’ antibodies; B) D-IHC control 5 performed with omission of the anti-CK primary antibody to detect cross-reaction between the two staining sequences; C) D-IHC control 6 without omission of any reactive. D-F display roughly equivalent areas of the JELN medulla: D) D-IHC control 4 performed with omission of both primary’ antibodies; E) D-IHC control 5 performed with omission of the anti-cytokeratin primary antibody to detect cross-reaction between the two staining sequences; F) D-IHC control 6 without omission of any reactives. The standard D-IHC demonstrate the correct functioning of the protocol used to carry out the D-IHC. Double- Iba1/CK cells (dark blue) as well as some single-Iba1 (brown cells) and single-CK (bright blue) (Images C and F) can be observed. Omission of anti-CK primary antibody reveals absence of non-specific binding for the second staining sequence of the protocol (images B and E). Omission of both primary antibodies demonstrates absence of non-specific binding for the whole procedure (images A and D). The bars represent 50 microns and sections were examined at 200X magnification. The C in red points to the capsule. For controls 4, 5 and 6 (Table 1).\u003c/p\u003e","description":"","filename":"additionalfile5.tif","url":"https://assets-eu.researchsquare.com/files/rs-3982091/v1/c0b345c2701ddc97573074e7.tif"},{"id":56237414,"identity":"0f2aa4e7-a82e-4c13-8901-aac24dcfcd78","added_by":"auto","created_at":"2024-05-10 09:04:45","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":14866504,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional File 6.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of cytokeratin (CK) and Iba1 expression in distal jejunum (DJE) of one cow with diffuse multibacillary lesions by double-immunohistochemistry (D-IHC).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA-C show roughly equivalent villi apical areas of the DJE where formation of granulomas is more frequent: A) D-IHC control 4 performed with omission of both primary’ antibodies; B) D-IHC control 5 performed with omission of the anti-cytokeratin primary antibody to detect cross-reaction between the two staining sequences; and C) D-IHC control 6 without omission of any reactives. D-F display equivalent basal areas of the lamina propia of the DJE: D) D-IHC control 4 performed with omission of both primary’ antibodies; E) D-IHC control 5 performed with omission of the anti-CK primary antibody to detect cross-reaction between the two staining sequences; F) standard procedure of D-IHC control 6 without omission of any reactives. Strong double- Iba1/CK cells (dark blue) as well as some single-Iba1 (brown cells) and single- CK (bright blue) (Images C and F) can be observed; some single-CK light blue immunostaining out of the granulomas that may seem unspecific is observed in the villi but this staining is part of the normal and known expression of CK in the intestine. Omission of anti-cytokeratin primary antibody reveals absence of non-specific binding for the second staining sequence of the protocol (images B and E). Omission of both primary antibodies demonstrates the absence of non-specific binding for the whole procedure. The bars represent 50 microns and sections were examined at 200X magnification. For controls 4, 5 and 6 (Table 1).\u003c/p\u003e","description":"","filename":"Additionalfile6.tif","url":"https://assets-eu.researchsquare.com/files/rs-3982091/v1/e75ded9cdd947952949b5fa7.tif"},{"id":56237408,"identity":"1701ead0-eeeb-4b7b-a993-2c14abe5c189","added_by":"auto","created_at":"2024-05-10 09:04:41","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":5354894,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional File 7\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSchematic representation of the structure and cellular components of the two distribution patterns of double Iba1/CK- positive cells (EMs) observed in the granuloma of Map infected animals with different pathological forms of PTB.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) Granuloma of multifocal animals and B) Granuloma of diffuse animals. Schemes generated by BioRender.com (2020). Retrieved from \u003ca href=\"https://app.biorender.com/biorender-templates\"\u003ehttps://app.biorender.com/biorender-templates\u003c/a\u003e\u003c/p\u003e","description":"","filename":"Additioanalfile7.tif","url":"https://assets-eu.researchsquare.com/files/rs-3982091/v1/c1b8a04d0e89dc3c4c9834fa.tif"},{"id":56237406,"identity":"ee3bab8b-71ae-4dd0-9b08-96993f8f7856","added_by":"auto","created_at":"2024-05-10 09:04:40","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":124821,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-3982091/v1/6bb883d4d62ce3d6eaaecd0c.docx"}],"financialInterests":"","formattedTitle":"Cytokeratin expression and distribution pattern of epithelioid macrophages in granulomatous lesions of animals with different pathological forms of bovine paratuberculosis: cytokeratin as a biomarker of resilience.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBovine paratuberculosis (PTB) is a chronic granulomatous enteritis caused by \u003cem\u003eMycobacterium avium\u003c/em\u003e subsp. \u003cem\u003eparatuberculosis\u003c/em\u003e (Map) responsible of important economic losses in the dairy industry. Map has been associated with several inflammatory and autoimmune diseases in humans such as Crohn\u0026rsquo;s disease [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], rheumatoid arthritis [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], type I diabetes [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], or multiple sclerosis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. PTB is listed by the World Organization for Animal Health (OIE) as a noticeable disease [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] due to its potential public health implications and its direct effects on animal health, economic losses, and livestock trade.\u003c/p\u003e \u003cp\u003eDepending on the severity of the clinical signs observed, the potential for shedding bacteria into the environment, and the ease with which the disease may be detected by current laboratory methods, four stages of PTB are defined [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]: silent, subclinical, clinical, and advanced clinical. In Map infected animals, a variety of granulomatous lesions associated with the different clinical stages of the disease can be detected, which have been classified in three main types: focal, multifocal, and diffuse [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Focal lesions consist of small scattered and well-demarcated granulomas composed by macrophages and few Langhans giant cells, mainly located in the jejunal (JELN) and ileal lymph nodes (ILN) and not affecting the intestinal lamina propria. Multifocal lesions consist of numerous well-demarcated granulomas in the intestinal lymphoid tissues and in the intestinal lamina propria. Diffuse lesions are characterized by extensive severe inflammatory infiltrate with granulomas in the intestinal lymphoid tissues and lamina propria, that markedly alter the normal histological structure of the intestine including submucosa, resulting in a clear merger of the villi. According to the inflammatory cell type present in the infiltrate and the number of acid-fast bacilli, diffuse lesions are further subdivided into diffuse lymphoplasmacytic paucibacillary, diffuse intermediate, and diffuse histiocytic multibacillary lesions [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMap infection produces a chronic and granulomatous inflammatory response characterized by the presence of granulomas, host-derived immune structures that consist of mononuclear cells that differentiate into macrophages, epithelioid cells, and multinucleated giant cells. Macrophages forming the granulomas are at different activation stages, which may or may not be associated with other inflammatory cell types [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Macrophages are phagocytic cells that play a central role in the defence of the host, as part of the host's innate immune response to Map infection [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Map is engulfed by intestinal macrophages as part of the initial response to infection, however, Map can disrupt normal macrophage functions and avoid destruction by preventing phagolysosome fusion, acidification, and activation [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Map can hide within infected macrophages during the long subclinical stage of PTB. Only a small percentage of the cattle exposed to Map developed clinical [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. There are other factors such as host genetics, environmental conditions and the infectious dose that also determine the animal\u0026rsquo;s ability to control MAP infection.\u003c/p\u003e \u003cp\u003ePTB progression results in various changes of the macrophages within the granuloma [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], resulting in a range of histological appearances [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. One of these changes is the epithelioid transformation of macrophages which produces cells with flattened shape, ovoid nuclei, and membranes that interdigitate with adjacent cells leading to the denomination epithelioid macrophages (EMs), also known as epithelioid cells. Although these cells are macrophage-derived, EMs are regarded as a very specialized type of mononuclear phagocyte immobilized in the granuloma, whose function has changed from phagocytosis to extracellular secretion [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Epithelial macrophages express at least one canonical epithelial marker. Cytokeratin (CK), a cytoskeletal protein highly specific of epithelial cells [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], has been previously used as a marker of epithelioid macrophages during the evolution of chronic granulomatous inflammation in a teleost fish model (pacus, \u003cem\u003ePiaractus mesopatamicus\u003c/em\u003e) induced by Bacillus Calmette-Guerin (BCG) demonstrating that macrophages with phagocytic activity became transformed into epithelioid cells with secretory activity [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. CK has also been used as an epithelioid cell marker in the ulcerative mycosis granulomas of Atlantic menhaden [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe associations between host genetics and the different PTB-associated lesions have been studied [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] identifying 192 and 92 single nucleotide polymorphisms (SNPs) that define 13 and 9 quantitative trait loci (QLTs) associated with multifocal and diffuse lesions, respectively. They suggest that distinct genetic variants might control the multifocal and diffuse lesions as no overlap was seen in the SNPs associated with each type of these lesions which could represent divergent disease outcomes. Pathway analysis with candidate genes overlapping the identified QLTs revealed a significant enrichment of the keratinization pathway in the animals with multifocal lesions. Gene members of the keratin (KRT) family such as KRT5, KRT7, KRT72, KRT73, KRT74, KRT75, KRT80, KRT81, and KRT83 were associated with the multifocal lesions. KRTs belong to the largest subgroup of intermediate filament (IF) family of cytoskeletal proteins and represent the most abundant proteins in epithelial cells, where they play a major role protecting epithelial cells from cell death, repairing cells and maintaining the stability and integrity of the gastrointestinal epithelium. They carry out other functions being, for example, responsible for the response and adaptation to various stresses, as conveyed by the broad array of crippling clinical disorders caused by inherited mutations in KRT coding sequences [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. For instance, expression of KRT5 in distal airway stem cells is essential for lung regeneration after H1N1 influenza virus infection [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], KRT7 may play a role in the histogenesis of small intestinal carcinoma associated with Crohn's disease [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and the KRT75 gene has been shown to be a candidate gene associated with heat stress adaptation in Chinese cattle [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Thus, CK expression could contribute to the containment of Map infection in several ways. Canive et al. (2021) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] hypothesized that the overexpression of CK in animals with multifocal lesions could provide tissue resilience through formation of epithelioid granulomas. The function of granuloma and the consequences of epithelioid transformation in granulomas caused by mycobacterial infections are not well understood. Thus, the starting hypothesis of the work presented here is that genetic variants of the keratinization pathway may predispose cattle to develop multifocal lesions able to prevent Map dissemination and limit tissue damage through formation of ordered granulomas where EMs, secretory cells that express CK, have a relevant role in controlling infection. In this context, the aim of the present study was to confirm, at the protein level, the enrichment of the keratinization pathway in cattle with multifocal lesions, and to investigate whether the presence of multifocally distributed epithelioid granulomas leads to the control of Map infection. For this purpose, the number and distribution pattern of EMs in distal jejunum (DJE) and JELN granulomas of animals with different pathological forms associated with PTB (focal, multifocal, diffuse intermediate, and diffuse multibacillary) and in negative control animals without lesions was investigated through quantitative double-immunohistochemical (D-IHC) analysis (using ionized calcium-binding adapter molecule-1 (Iba1) and CK as markers of macrophages and epithelial cells, respectively.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals and Samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCattle were selected from two farms located in the Principality of Asturias (Northwest of Spain). A total of 32 Holstein Friesian cows (0.81-10.39 years) were used for this study. Samples of blood, faeces and tissues were collected from all the animals. Blood samples were taken in serum clot activator Vacutainer® tubes (Vacuette, Kremsmunster, Austria) from the coccygeal vein and then transported to our laboratory at room temperature (RT). Serum was separated by centrifugation (2500 x g for 20 min at RT), Samples of faeces were collected, aliquoted and then stored at −20°C for subsequent ELISA analysis. Tissue samples (DJE, ileocecal valve (ICV), and ILN and JELN) were collected from the slaughtered animals \u003cem\u003ein situ\u003c/em\u003e at the local abattoir after evisceration and they were used for histological classification and D-IHC (DJE and JELN). The Map infection status of the 32 animals used in this study was determined by histopathology, specific antibody serum ELISA test (IDEXX, Montpellier, France), bacteriological culture, and specific real-time PCR of tissues and faeces, following the procedures previously described [26].\u0026nbsp;As an indirect estimation of the amount of Map present in each histopathological group (our gold standard) we created an infection score assuming that the amount of Map present in an animal is related to the positivity found in ZN, ELISA, PCR and the bacteriological culture. The mean infection score of each histopathological group was calculated as the sum of the total number of positive diagnosis test (counting 1 for each positive, 0.5 for each non-conclusive result and 0 for each negative result) plus the total number of ZN positive results (1 if there is one + in the ZN result, 2 for two ++ and 3 for +++) split by the total number of animals in the group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissue preparation and histopathological classification of animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissue samples were taken and processed using standard procedures. \u0026nbsp;Samples were fixed in 10% neutral buffered formalin, sliced, and embedded in paraffin blocks. Tissue sections (4 µm) were cut and placed on microscope slides (Superfrost Plus, Menzel GmbH, Braunschweig, Germany), and dried at 37\u003csup\u003e°\u003c/sup\u003eC for 24 h. Afterwards, tissue sections were stained by hematoxylin-eosin (HE) and Ziehl-Neelsen (ZN) to evaluate lesions and confirm the presence of acid-fast bacteria. Slices were analysed using an Olympus BH-2 light microscope (Olympus, Tokyo, Japan). Pathological lesions associated with bovine PTB were classified according to Gonzalez et al. (2005). The classification was carried out by examining four complementary target sections of the gut tissue (DJE, ICV, ILN and JELN).\u0026nbsp;Once examined the four sections for all the animals (focal, multifocal, and diffuse) included in the study, DJE and JELN were selected to carry out the D-IHC as these two sections showed overall more abundant granulomas. In multifocal animals, the main target group under study, most granulomas were present in DJE (the number of granulomas in ICV was lower) and JELN. Focal animals did not often present granulomas in DJE lymphoid tissue, so examination of\u0026nbsp;JELN sections was important to the experiment. Diffuse lesions were observed in the four sections. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDouble-immunohistochemistry (D-IHC) \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor quantification of the number of CK-positive macrophages (EMs) within granulomas of animals with different pathological forms associated with PTB (focal, multifocal, diffuse intermediate, and diffuse multibacillary) and in negative control animals without lesions the number of cells expressing CK and Iba1 within the DJE and JELN of each animal was investigated by sequential double-immunohistochemistry\u0026nbsp;\u003cem\u003eto detect two different antigens Iba1 and CK in tissue samples\u003c/em\u003e (Additional File 1). Formalin-fixed paraffin-embedded DJE, and JELN samples were cut into 3-µm sections and placed on microscope slides (Superfrost Plus, Menzel GmbH, Braunschweig, Germany). Sections were dewaxed and rehydrated using tap water RT. Antigen retrieval was performed using 0.1% Trypsin (Sigma-Aldrich, St. Louis, MO, USA) dissolved in preheated Tris-buffered saline (TBS) containing 0.1 % CaCl\u003csub\u003e2\u003c/sub\u003e (Merck, Darmstadt, Germany) for 45 min at 37\u003csup\u003e◦\u0026nbsp;\u003c/sup\u003eC. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide (Sigma-Aldrich, St. Louis, MO, USA) in methanol (VWR, Monroeville, PA, USA), 10 min at RT. Slides were washed with tap water at RT, and then non-specific binding was blocked using 10% normal goat serum (Vector Laboratories) containing 3% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA) for 15 min at RT.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor the first stain, tissue sections were incubated with rabbit polyclonal antibody anti-Iba1 (FUJIFILM Wako, Osaka, Japan) for specific macrophage identification (Iba1 is largely restricted to cells of monocyte/macrophage lineage [27] at a 1:1200 dilution overnight at 4 °C and then washed three times with TBS 1X (1X TBS, 5 mM Tris (Merck KGaA, Darmstadt, Germany)/HCl (Panreac Química, SLU, Barcelona, Spain) pH 7.6, 136 mM NaCl (Merck KGaA, Darmstadt, Germany)) at RT. After that, sections were incubated for 30 min at RT with a biotinylated anti-rabbit IgG secondary antibody produced in goat (Vector Laboratories, Burlingame, CA, USA) at 1:200 dilution and slides washed as previously described. For signal detection sections were incubated for 30 min at RT with Avidin-Biotin Complex (ABC kit Peroxidase (PO) Standard, Vector Laboratories) followed by three washes with 1X TBS and incubation with 3,3`-Diaminobenzidine tetrahydrochloride (DAB) (Sigmafast, Sigma-Aldrich, St. Louis, MO, USA) for 2 min at RT. Afterwards, samples were rinsed with tap water for 5 min and washed with 1X TBS three times at RT. It is important to carry out the double-staining procedure in the specified order, using as first substrate the DAB. This sequence was previously recommended by other authors, because the DAB reaction product, a widely recognized chromogen, effectively masks and prevents unspecific cross-reactions [28]. Then, for the second D-IHC stain, the same slides were incubated overnight at 4 °C with rabbit anti-cytokeratin polyclonal (Dako, California, USA) at a 1:1000 dilution. It is a wide-spectrum screening antibody that detects low-molecular-weight CKs (40-54 kDa), specifically KRT7-8 and KRT17-20 according to Moll’s designation, and high-molecular-weight CKs (48-67 kDa), specifically KRT1-6 and KRT9-16. Samples were washed three times with 1X TBS at RT. Bounded antibody was detected by incubation for 30 min at RT with alkaline phosphatase (AP)-conjugated goat anti-rabbit IgG secondary antibody (Sigma-Aldrich, St. Louis, MO, USA) followed by three washes with 1X TBS and 1-StepTM NBT/BCIP (Thermo Scientific, Rockford, USA) for 6 min at RT under microscopic control. There was no need to block endogenous AP activity. Samples were rinsed with tap water for 5 min at RT and counterstained in Mayer’s hematoxylin (MerckKGaA, Darmstadt, Germany) for 5 seconds before washing, dehydrating, and mounting with DPX (Merck KGaA, Darmstadt, Germany). Single-Iba1 positive cells stained brown (DAB), single-CK positive cells stained blue (1-StepTM NBT/BCIP), and double Iba1/CK-positive cells stained dark blue or black. Because the AP substrate (1-StepTM NBT/BCIP) and Avidin-Biotin PO substrate (DAB) can produce a widespread precipitate over the entire section, both reagents were freshly prepared and micropore filtered 0.22 µm (Merck Millipore, Cork, Ireland) immediately before application to the tissue. Prior to the D-IHC optimization, a single immunohistochemistry (S-IHC) experiment was carried out to evaluate positive CK staining in the experimental samples (DJE and JELN) using as a CK-positive epithelial control tissue bovine endometrium [29,30] (Table 1). In bovine endometrium, a positive control (Control 1) without omission of any reactive was carried out, and a negative control (Control 2) performed with omission of primary antibody. Moreover, negative controls (Control 3) performed with omission of anti-CK primary antibody were analysed in experimental samples from animals with different types of PTB-associated lesions. Two negative controls and one positive were included in the D-IHC experiment (see Table 1). The first negative control (Control 4) consisted of slides processed as previously described for D-IHC with omission of the two primary antibodies used to detect unspecific binding of the secondary antibodies to the tissue, the second negative control (Control 5) consisted of slides treated as previously described with omission of the anti-CK primary antibody to detect cross-reaction between the two staining steps, and the positive control (Control 6) was performed without omission of any primary or secondary antibody.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImage acquisition and quantification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunolabelled sections from D-IHC were observed using an Olympus BH-2 light microscope (Olympus, Tokyo, Japan). Individual images were acquired using an Olympus DP-12 digital camera (Olympus, Tokyo, Japan). Images were taken throughout histological sections selecting fields with granulomatous lesions avoiding areas containing preparation artefacts, cell debris, or the edges of the slide. In control animals, although granulomas are not present, counting was carried out in equivalent regions of the preparations, in other words, the images were taken in areas where granuloma formation usually occurs, such as the cortical and paracortical area in the JELN and the apical area of the villi in the DJE or more precisely on villous tips.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo evaluate cytokeratin expression in cattle with different histopathological forms of PTB, the number of single and double positive stained cells within granulomatous lesions and controls in each tissue sample from each individual was counted in ten randomly selected fields per individual at a final magnification of 400X so a total of 50-70 fields were examined for each histopathological type. Each field corresponded to one image captured using a 40X objective (1600 x 1200 pixels) having a\u0026nbsp;theoretical area of 41207.52 µm2 (234,4 µm length x 175,8 µm height).\u0026nbsp; The number of positive immunolabelled cells was manually counted in each selected field/image using the ImageJ program (National Institutes of Health, EEUU).\u0026nbsp;Results are expressed as a mean of the number of positive cells per field ± standard deviation (SD) for each histopathological group.\u0026nbsp;Positive cells have been classified into single-CK positive cells, single-Iba1 positive cells and double-Iba1/CK positive cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis was carried out using the R program (R Development Core Team, version 4.1.3). Differences of quantitative variables between two groups was carried out using the Student´s t-test or Wilcoxon test for independent samples, depending on normality hypothesis. Differences between three or more groups were analysed using the ANOVA or Kruskal-Wallis´s test, depending on whether the hypotheses of homoscedasticity and normality were verified. When the Kruskal-Wallis´s test and the ANOVA´s tests were statically significant the non-parametric post hoc Dunn´s test and the parametric post hoc Tukey´s test, respectively, were conducted to determine exactly which pairwise groups had statically significant differences. The level of significance used was 0.05. \u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eMap infection status of studied animals\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Map infection status of the 32 animals used in this study is shown in Table 2. \u0026nbsp;The animals were classified into five groups according to the presence/absence and type of histological lesions in gut tissues (Gonzalez et al., 2005) (see Table 2): \u0026nbsp;1) the healthy control group (n=5), which consisted of animals with no observed PTB-associated lesions, negative by ELISA, and bacteriological culture and PCR of tissues and faeces; 2) the focal group (n=7) consisted of animals with focal lesions, all ZN positive and ELISA negative; 3) the multifocal group (n=7) included animals with PTB-associated multifocal lesions, positive by at least one of the diagnostic tests used. In this group, there was one animal, the only one showing clinical signs that was \u0026nbsp;ZN+ \u0026nbsp; but \u0026nbsp;curiously it was negative by the rest of the tests; 4) the diffuse intermediate group (n=7) included animals with PTB-associated diffuse intermediate lesions with most animals positive by ELISA and PCR of faeces and tissues, and 71% of the animals showing clinical signs; and 5) the multibacillary diffuse group (n=6), which was composed of animals with multibacillary diffuse lesions with a large number of Map bacteria present, with 100% of the animals positive by ELISA and with PTB-associated clinical signs. Clinical signs were observed in 0%, 20 % and 85% of the animals with focal, multifocal and diffuse (intermediate and multibacillary) lesions (no information was available for 3 focal and 2 multifocal animals). No gross or histologic lesions compatible with other inflammatory processes were identified during post-mortem inspection of the studied animals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of the\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ecross-reactivity and specificity of the\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eimmunoreagents used in the single and double-immunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePositive CK staining was first evaluated by S-IHC in bovine endometrium used as a positive control for epithelial CK expression and in JELN and DJE of animals with different pathological forms of PTB (Table 1, Controls 1 to 3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn bovine endometrium, the positive control (Control 1), carried out without omission of any reactive, CK staining was observed exclusively in the cytoplasm\u0026nbsp;of epithelial cells of the luminal epithelium, superficial glands within the stroma and \u003cem\u003eStratum basalis\u0026nbsp;\u003c/em\u003edeep glands (Additional File 2A-C);\u0026nbsp;the negative control (Control 2), performed with omission of the anti-CK primary antibody, no immunolabelled cells were observed (Additional File 2D-F). The anti-CK wide spectrum screening antibody used exhibited specificity in the positive bovine endometrium control and the biotinylated anti-rabbit IgG secondary antibody displayed no perceptible cross-reactivity with non-target proteins.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn JELN and DJE samples CK staining was observed in all animals. In JELN (Additional File 3), positive cells were observed in the cortex and close to or forming part of granulomas (Additional File 3A-D), near connective tissue and trabeculae (Additional File 3E-H), and in the medullar area (Additional File 3I-L) for all type of PTB lesions. The number of CK-positive cells seemed to be more abundant toward the efferent lymphatic vessels (Additional File 3A, 3E, 3I for animals without lesions, 3B, 3F, 3J for focal animals, 3C, 3G, 3K for multifocal animals and 3D, 3H, 3L for intermediate diffuse animals). As the severity of PTB lesions increases so does the number of CK-positive cells, especially numerous in multifocal lesions (Additional File 3A-D in cortex, 3E-H in areas close to connective tissue and trabeculae, 3I-L in medullar area). In DJE (Additional File 4), CK-positive cells were detected in all animals scattered in the mucosa of DJE (Additional File 4A, 4E, 4I for animals without lesions, 4B, 4F, 4J for focal animals, 4C, 4G, 4K for multifocal animals and 4D, 4H, 4L for intermediate diffuse animals), throughout the lamina propria in the apical area (Additional File 4A-D and 4E-H), forming part of the granulomas (Additional File 4G and 4H), and around and in the crypts of Lieberkühn (Additional File 4I-L). As in the case of JELN samples, it appears that as the severity of the PTB-associated lesions increased, CK-positive cells became more numerous, especially in multifocal lesions (Additional File 4A-D in apical area, 4E-H in apical granulomas or equivalent area and 4I-L in basal area of DJE). \u0026nbsp;No staining was observed when the anti-CK antibody was omitted (Control 3 of Table 1, results not shown).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCross-reactivity and specificity of detection antibodies in the D-IHC of JELN and DJE tissue samples was assessed analysing three controls (Table 1, D-IHC controls 4 to 6). In this assay, we expected to observe three types of positive immunolabeled cells (single-CK (light blue), single-Iba1 (brown) and double-Iba1/CK (dark blue) positive cells) and negative immunolabelled cells. In the negative control (Control 4) performed with omission of the two primary antibodies (anti-Iba1 and anti-CK) no positive cells were observed (Additional Files 5A and 5D for JELN and 6A and 6D for DJE) indicating that secondary antibodies displayed imperceptible cross-reactivity with non-target proteins and that PO and AP substrates did not show unspecific reactivity. In the negative control carried out with omission of the anti-CK primary antibody (Control 5) no single-CK (light blue) or double-Iba1/CK positive cells (dark blue) were observed (Additional Files 5B and 5E for JELN and 6B and 6E for DJE) indicating that in the sequential D-IHC procedure design the goat anti-rabbit IgG AP-conjugated secondary antibody did not bind unspecifically to anti-Iba1 primary antibodies; no cross-reactivity was detected between the two staining sequences. In the\u0026nbsp;D-IHC\u0026nbsp;positive control (Control 6),\u0026nbsp;carried out without omission of any reactive,\u0026nbsp;the three types of positive cells were observed within the granuloma (Additional Files 5C and 5F for JELN and Additional Files 6C and 6F for DJE). \u0026nbsp;In the DJE positive control, some single-CK light blue immunostaining was observed out of the granulomas in the villi as part of the normal expression of CK in the intestine. These results demonstrate that the procedure of the D-IHC worked well in both tissue types, with no cross-reactivity observed between detection antibodies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorphological analysis, distribution, and patterns of Iba1 and cytokeratin expressing cells in jejunal lymph nodes and distal jejunum of cattle with different histopathological forms of bovine paratuberculosis.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe enrichment of the keratinization pathway in cattle with PTB-associated multifocal lesions was investigated at the protein level \u003cstrong\u003ewithin granulomas\u003c/strong\u003e of JELN and DJE samples, by double-Iba1/CK immunohistochemistry and quantification of the number of CK-positive EMs in animals with different pathological forms of PTB (n=25) and control animals without lesions (n=5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eD-IHC\u0026nbsp;analysis showed four types of cells within the granuloma and around the structure of both JELN and DJE samples (Figures 1 and 2, respectively): single-Iba1 positive macrophages stained as brown cells (Figure 1A for JELN and 2A for DJE), single-CK positive cells detected as bright blue cells (Figure 1B for JELN and 2B for DJE), double-Iba1/CK positive macrophages expressing CK observed as dark blue or black cells (Figure 1C for JELN and 2C for DJE), and negative cells (no immunolabelled cells).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn JELNs (Figure 1 and 3), both expected CK-positive cell types (single-CK and double-Iba1/CK positive cells) were observed in all the samples (N=32), including the control samples, with no morphological differences observed in these cell types between animals of different groups and ages. Single-CK immunolabelled cells (bright blue) in the JELN were observed in a highly variable number as part of the granuloma and scattered in the cortex (Figure 1D) and sometimes near blood vessels, supporting tissue and trabeculae (TB) (Figure 1E) and medullar area (Figure 1F). Single-CK positive cells had medium to large-sized nuclei (4.22-8.65 µm in diameter) with a semi-round shape and sparse cytoplasm compatible with reticular cells (RCs) (similar morphology and localization) (Figure 1B). \u0026nbsp;Double-Iba1/CK positive cells had large round to oval nuclei (4.36-9.12 µm in diameter) and abundant cytoplasm (Figure 1C). These double-immunolabeled cells were in the cortex of the lymph nodes in a scattered manner (Figure 1D), around the lymphoid follicles, as well as close to the germinal centre and in the subcapsular sinus. These cells were also observed in the paracortex area and especially in the medullary area tending to be more numerous toward the efferent lymphatic vessels in both infected and uninfected animals (Additional File 5C and 5F).\u0026nbsp;Control animals without lesions showed a similar pattern of CK-expression to that mentioned above (Figure 3A and 3B), however\u0026nbsp;as the severity of the PTB-associated lesions increases, the number of double-Iba1/CK positive cells increases in the cortex area, showing two different patterns, either double-Iba1/CK positive cells were found surrounding the granuloma or they were within the granuloma forming the granuloma itself (Additional File 7). The first scenario was more common in animals with focal and multifocal lesions (Figure 3C, 3D, 3E and 3F) while the second one seemed to be more frequent in animals with diffuse intermediate and diffuse multibacillary lesions (Figure 3G, 3H, 3I and 3J). These two patterns could be also observed in single CK-immunochemistry (See Additional File 3) confirming the results of the D-IHC. Multinucleated giant cells, both CK-positive (Figure 3G and 3I) and CK-negative, were also observed in JELN. With respect to single-Iba1 positive macrophages (stained as brown cells), these cells present large round to oval nuclei (5.11-9.83\u0026nbsp;µm in diameter) and abundant cytoplasm\u0026nbsp;(Figure 1A) with a similar appearance to tissue macrophages that had the expected distribution.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn DJE (Figure 2 and 4), single-Iba1, single-CK and double-Iba1/CK positive cells were also observed in all animals. Single CK-stained cells had medium- to large -sized round nuclei with scarce cytoplasm (Figure 2B). \u0026nbsp;These cells were observed in a highly variable number as part of the granulomas and scattered in the mucosa (Figure 2D-E) and submucosa (Figure 2F) of DJE Double-Iba1/CK positive, with large round to oval nuclei and abundant cytoplasm (Figure 2C), were located scattered throughout the lamina propria mostly in the apical area (Figure 2D), around and in the crypts of Lieberkühn (Figure 2E), surrounding the villi (Figure 4B, 4C and 4D). As in the case of JELN samples, control animals without lesions did not show a striking pattern of CK expression (Figure 4A and 4B), but it appeared that as the severity of the PTB-associated lesion increased so did the number of double-Iba1/CK positive cells in the granuloma area, the granulomas showing the same two patterns previously described (Figure 4F where double-Iba1/CK positive cells are found surrounding the granuloma and 4H where they were within the granuloma). These two patterns could be also observed in single CK-immunochemistry (See Additional File 4G and 4H).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of cytokeratin expression at the protein level in granulomatous lesions of cattle with different histopathological forms of bovine paratuberculosis.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results of the quantification of the number of single and total-Iba1, single and total-CK and double-Iba1/CK positive cells in \u003cstrong\u003eJELN\u003c/strong\u003e are shown in Table 3.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWith respect to the number of single-CK positive cells (cells expressing cytokeratin that are not apparently macrophages) per field and histological group,\u0026nbsp;significant differences were observed between the different histopathological groups (Kruskal-Wallis test, p \u0026lt;0.001).\u0026nbsp;Specifically, post hoc Dunn´s test reported differences between the medians of the multifocal group (5.00 (2.00-14.00)) with the focal (3.50 (0.25-5.55)) and the control (2.00 (0.00-6.00)) groups (p=0.015 and 0.002, respectively). Regarding the total number of macrophages (single-Iba1 cells) no significant differences were observed between groups (Krustal-Wallis, p-value=0.504).\u003c/p\u003e\n\u003cp\u003eAs for the mean number of double-immunolabelled cells (Iba1 cells expressing CK, EMs) per field the group of animals with multifocal lesions showed the highest mean number (51.59 ± 24.40) and the control group the lowest (29.64 ± 20.52). In this case, significant differences between the cows with multifocal lesions and the cows with focal, diffuse intermediate, diffuse lesions (including intermediate and multibacillary) and controls (Tukey´s test, p\u0026lt;0.01, p\u0026lt;0.01, p=0.005 and p\u0026lt;0.01, respectively). The multifocal group had higher numbers of double-immunolabeled cells than the diffuse multibacillary group, however, no significant differences were observed (p=0.165). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs for the total number of CK-expressing cells (single and double) and in comparison, with the number of single-CK-expressing cells, the main difference is that when we analysed the total number significant differences of the multifocal with the diffuse group (p=0.023) were now observed. Overall, the significance level of the differences observed between groups was higher when we compared numbers of double-Iba1/CK immunolabelled cells. No significant differences were observed in the total number (single and double) of Iba1 positive cells between groups (ANOVA, p-value=0.185).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSignificant differences in the mean values of single-CK, double-Iba1/CK and total CK- expressing cells were also observed between infected animals with any type of lesions (focal, multifocal and diffuse animals) and control animals without lesions (Welch´s test p\u0026lt;0.001, Student´s test p=0.001 and Welch´s test p\u0026lt;0.001, respectively), showing the control animals lower mean numbers than the infected animals with any type of lesion.\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe levels of cytokeratin expression in granulomas of cattle with different histopathological forms of bovine paratuberculosis was also evaluated in \u003cstrong\u003eDJE\u003c/strong\u003e samples (Table 4). Regarding single-CK positive cells, significant differences were found between infected animals (focal, multifocal and diffuse) and control animals (Test of Welch, p-value=0.045) and between the multifocal and the focal groups (Dunn´s test, p-value=0.002), showing the multifocal group the highest numbers of immunolabelled cells. No significant differences between pairs of histopathological groups were observed for single-Iba1 positive cells, double-immunolabeled cells and total number of CK-immunolabeled cells. Significant differences were detected in the total number of Iba1-positive cells between the multifocal and the diffuse intermediate groups. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn order to shed light on the consequences of epithelioid transformation of macrophages in Map infection and disease progression the ratios of EMs (double Iba1/CK positive cells) with respect to non-epithelioid macrophages (non-EMs) (single-Iba1 positive cells) and the total number of macrophages (single and double-Iba1 cells) was also investigated (Table 5). In JELN it was found that the ratios of EMs/non-EMs were lower than one (0.39-0.75) indicating that the number of non-EMs (single Iba1-positive cells) was higher than the EMs, with multifocal animals showing the highest ratio of EMs/non-EMs (higher numbers of EMs than the rest of the histopathological groups). Likewise, infected animals showed higher ratios than control animals. In DJE, all the histopathological groups present similar ratios of EMs/non-EMs, which were close to 1 indicating that they had similar amounts of EMs and non-EMs. In DJE lower total numbers of non-EMs and higher of EMs than in JELN were observed.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe knowledge of the composition and structure of the granuloma, made up of macrophages in different stages of differentiation and other immune cells, may help to understand the role of the granuloma in the control of Map infection.\u003c/p\u003e \u003cp\u003eIn the present study, the number and distribution pattern of CK-expressing cells in the granulomas of animals with different pathological forms of bovine PTB was analyzed for the first time to investigate the role of epithelioid granulomas in the control of Map infection. Our results show that CK expression was enriched in granulomas of cattle with multifocal lesions. Multifocal animals had significantly higher numbers of double CK/Iba1 positive EMs in JELN granulomas (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and higher numbers of single-CK expressing cells in JELN and DJE granulomas (Tables\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Likewise, infected animals had significantly higher levels of single and double-CK positive cells than control animals. In DJE, no significant differences in the number of EMs were observed between histopathological groups or between infected and non-infected control animals. However, significant differences in the number of single-CK cells were observed between infected and non-infected animals. The number of macrophages in the control group without lesions it is quite high in comparison with the rest of the evaluated histopathological groups. This is expected as the gastrointestinal system has the largest number of tissue macrophages in the intestine [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The high number of EMs could be explained because in natural infections animals may be exposed to other immunological stimuli, which may affect epithelioid transformation of macrophages [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Regarding EMs, two different distribution patterns were observed (Additional File 7) in JELN and DJE. Granulomas of multifocal animals presented an ordered structure where EMs were forming a barrier surrounding the granuloma. Moreover, multifocal animals had significantly lower infection scores than diffuse animals and most animals were subclinical. These findings suggest that this barrier is preventing dissemination of Map and maintaining tissue integrity, playing a relevant role in controlling infection. Animals with diffuse lesions, where CK-expressing EMs were observed throughout the granuloma and did not form a barrier around them, had higher titers of Map, showed clinical signs and were not able to contain the infection efficiently despite having high titers of anti-Map antibodies. Therefore, the number and especially the distribution pattern of EMs in the different histopathological groups had an effect of in the control of Map and in the development of clinical disease.\u003c/p\u003e \u003cp\u003eVarious hypotheses about the consequences of epithelioid transformation of macrophages have been postulated, some authors believe that it engenders highly phagocytic and microbicidal [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] while others suggest that it produces non-phagocytic cells with secretory functions that enhance the immune response [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. A longitudinal study [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] of tuberculous granulomas induced by chronic \u003cem\u003eM. marinum\u003c/em\u003e infection of frogs revealed that both types of epithelioid cells (phagocytic and microbicidal cells/ non-phagocytic cells with secretory functions) could be present in chronic granulomas, showing that, even long-term granulomas (long subclinical stage) are dynamic environments where bacterial replication and their phagocytic clearance maintain relatively stable bacterial numbers despite the established immune response. Even though the granuloma tends to become more epithelioid with time, even in the most chronic phases, infected and highly activated non-EMs were found together with epithelioid cells. In the present study, non-EMs and EMs were found in DJE and JELN showing similar numbers in DJE while higher numbers of non-EMs were observed in JELN. Further experiments should be performed to investigate the presence of Map in macrophages and the types and function of EMs (phagocytic and microbicidal cells/ non-phagocytic cells with secretory functions) in the granulomas of Map-infected animals. Tissue-resident macrophages are extremely heterogeneous in a manner that is suited to tissue-specific [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The differences observed between DJE and JELN could be due to the fact that DJE and JELN have different functions while one of the fundamental functions of DJE is tissue homeostasis involving macrophages that participate in tissue repair and resolution of inflammation the main function of JELN is immunosurveillance, the heterogeneity of macrophages due to these functions may result in changes in epithelioid differentiation processes. Moreover, gut macrophages in DJE have a half-life estimated of 3 weeks [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], which means that cells are in a continuous renewal, affecting the accumulation of epithelioid macrophages which might not allowed to detect differences between groups.\u003c/p\u003e \u003cp\u003eThe role of the granuloma in mycobacterial infections, such as tuberculosis, has been previously revised [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Historically, it has been regarded as a host-protective structure that walls off the infecting mycobacteria, constituting a barrier to bacterial proliferation and dissemination. In contrast, an opposing trend has emerged that presumes that the granuloma is a highly dynamic structure promoting bacterial proliferation [\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. I has been shown that mycobacterial granuloma formation in a Zebrafish-\u003cem\u003eMycobacterium marinum\u003c/em\u003e model is accompanied by macrophage induction of canonical epithelial molecules and structures [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. They demonstrated that disruption of macrophage epithelial protein biomarkers such as E-cadherin, resulted in disordered granuloma formation which facilitates and enhances immune access, resulting in a decrease of bacterial burden and increase host survival suggesting that the granuloma has a bacteria-protective role. They suggested that E-cadherin expressing macrophages could be acting as an access barrier to protect bacteria from the host immune system in a similar structure disposition to the one observed in the present study in animals with focal and multifocal histological lesions where EMs were found surrounding the granuloma. However, in our study (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), animals presenting this EMs disposition in the granuloma were subclinical and had low titers of Map indicating that the barrier seems to be controlling infection rather than producing a favorable niche for mycobacteria. In leprosy, it has been shown that the development of a robust immune response results in paucibacillary disease, where well-developed granulomas contain scant organisms, whereas in multibacillary leprosy with poor to no granuloma formation high bacterial growth is observed. However, paucibacillary leprosy sometimes causes severe morbidity, indicating that an overexuberant granulomatous response is not good either, while restricting bacteria also harms the host [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Curiously, animals with multibacillary leprosy, not able to control mycobacteria replication, present down-regulation of the expression of keratinocyte-associated genes (KRT14 and KRT5) [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In tuberculoid leprosy, the presence of epithelioid giant cells and the formation of granulomas leads to the control of \u003cem\u003eMycobacterium leprae\u003c/em\u003e replication and the containment of its spread, however, in lepromatous lesions phagocytic foamy macrophages are observed and are unable to control \u003cem\u003eM. leprae\u003c/em\u003e replication [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe presence of non-EMs (Iba1-positive cells) was also investigated in DJE and JELN granulomas of the different histopathological groups and no significant differences were observed between histopathological groups and between non-infected and control animals in the number of Iba1-positive cells. In contrast, Jenvey et al. (2019) [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] showed that the number of macrophages in the mid ileum increases with progression of infection even though a significant number of macrophages were not associated with Map. This finding suggests that although the infected animal recruits more macrophages to the site of infection, these increased numbers of macrophages are unable to clear Map, which is transmitted from macrophage to macrophage leading to further progression of the infection, and the development of clinical disease. Even though our results are different from those reported by Jenvey et al. (2019) [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], it must be noted that we have used samples from a different section of the gut tissue (frozen \u003cem\u003eversus (vs)\u003c/em\u003e fresh, mid ileal \u003cem\u003evs\u003c/em\u003e. DJE and JELN) as well as a different detection technique (immunohistochemistry \u003cem\u003evs\u003c/em\u003e immunofluorescence), primary antibody (anti-Iba1 \u003cem\u003evs\u003c/em\u003e anti-macrophage surface antigen (clone AM-3- K)) and quantification protocol (mean labelled area in mid ileal tissue section \u003cem\u003evs\u003c/em\u003e mean number of macrophages in granulomas per field; in our case counting is restricted to granulomas in contrast with counting the total tissue section as in Jenvey et al.) [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. It is possible that we are also counting other cells of the monocyte/macrophage lineage using anti-Iba1 antibody [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDuring maturation several additional cell types are recruited within the granuloma that can define the impact and function of the granuloma within the infection or disease. In this sense, we have observed the presence of single-CK positive cells in the granulomas that showed similar morphology and localization to RCs, which are easily identified using cytokeratin as a marker. Here, higher numbers of single-CK expressing cells were observed in JELN and DJE granulomas of multifocal animals. Previous studies in humans showed the presence and distribution of CK-immunoreactive reticular cells in normal and pathological human lymph node [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. RCs are normally present in lymphoid organs such as lymph nodes and play a crucial role in immune response to infection or inflammation [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Moreover, RCs can constrain an excessive immune response [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. RCs were shown to be involved in transplantation tolerance, cancer immunity and they are considered as a key factor in autoimmune diseases [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], which have been associated with Map infection. In this sense, RCs could be also helping to control Map infection, however, this is beyond the scope of the current study.\u003c/p\u003e \u003cp\u003eContinuous exposure of animals to Map results in a dynamic balance where infection never gets established or is controlled by an efficient innate immune response in an important part of the farm population while in other individuals infection progresses to subclinical delimited focal or multifocal forms and in a smaller fraction of the herd to diffuse lymphocytic (cellular or Th1 type) or non-lymphocytic (humoral immune response or Th2 type) forms that will result in clinical disease [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Cellular immunity may exert some control over the infection at early stages, but this appears to be very different between individual cows, with different consequences on shedding and the development of a humoral response over [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. It might be possible that one of the factors involved in this switch from a Th1 to a Th2 response is the activation stage and organization of macrophages in the granulomatous lesion itself. It is possible that an increase in the number of EMs located around the granuloma forming an epithelioid barrier might inhibit granuloma growth by disrupting the recruitment of newly activated macrophages to the structure hindering mycobacterial macrophage-to-macrophage spread and thus serving to reduce their intracellular niche, favouring equilibrium towards EMs and walling off the bacteria inside the granuloma.\u003c/p\u003e \u003cp\u003eIn summary, the number and distribution pattern/organization of EMs in the granuloma have a relevant role in controlling Map infection. Multifocal animals can control infection through formation of granulomas where EMs expressing CK, localized around the granuloma, form a barrier that limits/repairs tissue damage protecting animals from clinical disease. Our results agree with the findings of Canive et al. (2021) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] where a significant enrichment of the KRT pathway was observed, at the gene expression level, in animals with multifocal lesions suggesting that KRT genetic variants may predispose cattle to develop multifocal lesions that maintain tissue resilience through rearrangements in the keratin filaments. It might be considered that animals that form this type of granuloma, might be resilient to the disease, in terms of controlling the shift from subclinical to the clinical disease. Whether this control is long-lasting needs to be further investigated, since there might exist external factors that trigger the loss of this control at the granuloma level, and therefore the progression of the disease. Our findings suggest that cytokeratin could be considered as a potential biomarker of PTB resilience. Tolerance is defined as the mechanisms used by the host to reduce susceptibility to tissue damage and other fitness damages caused by pathogens or immune response, so, in this sense, cytokeratin could be considered specifically as a biomarker of tolerance.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimals used in this study had their origin in commercial farms. All farmers were informed about the study and gave their consent and approval for the use of samples in the present study. This study was carried out in accordance with Directive 2012/63/EU of the European Parliament. Experimental procedures were evaluated by the SERIDA Animal Ethics Committee board approval and authorized by the Regional Consejer\u0026iacute;a de Agroganader\u0026iacute;a y Recursos Aut\u0026oacute;ctonos del Principado de Asturias, Spain (authorization codes PROAE 29/2015 and PROAE 66/2019).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study will be available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone of the authors of this manuscript have any competing or financial interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study is part of the I+D+I project\u0026nbsp;(PID2021-122195OR-C22), which was funded by the MCIN/AEI /10.13039/501100011033 / FEDER, UE and by\u0026nbsp;regional funds\u0026nbsp;PCTI 2021\u0026ndash;2023 (GRUPIN: IDI2021-000102).\u0026nbsp;We acknowledge the National Institute for Agricultural Research (INIA) for the scholarships of Cristina Blanco V\u0026aacute;zquez (CPD2016.0142 funded by MCIN/AEI/10.13039/50110001033 and FSE) and Alejandra Isabel Navarro Le\u0026oacute;n (PRE20200-096451).\u003c/p\u003e\n\u003cp\u003eThe funding bodies have not been involved in the design of the study and collection, anlaysis, and interpretation of data and in writing the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026acute;s contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDesign of the study: RC and AINL. Perform experiments: AINV, MM, CBV, NI, and TI. Wrote the manuscript: RC and AINL. All authors have read and approved the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to acknowledge ASTEGA Veterinary Services for their collaboration in the sampling work and the daily work of SERIDA\u0026acute;s farm operators in the care and maintenance of animals. We gratefully acknowledge Kevin P. Dalton for proofreading the manuscript and the Scientific Services of the University of Oviedo. \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eFeller M, Huwiler K, Stephan R, Altpeter E, Shang A, Furrer H, Pfyffer GE, Jemmi T, Baumgartner A, Egger M (2007) Mycobacterium avium subspecies paratuberculosis and Crohn\u0026apos;s disease: a systematic review and meta-analysis. 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Vet Res 46(1):62\u003cstrong\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 5 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"paratuberculosis, cytokeratin, epithelioid macrophages, granuloma, resilience","lastPublishedDoi":"10.21203/rs.3.rs-3982091/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3982091/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA recent genome-wide association study identified 92 genetic variants in cattle with paratuberculosis (PTB)-associated multifocal lesions. Pathway analysis with the identified candidate genes revealed a significant enrichment of the keratinization (KRT) pathway in those animals. To confirm, at the protein level, this enrichment the number and distribution pattern of cytokeratin (CK)-expressing cells in granulomas of distal jejunum (DJE) and jejunal lymph nodes (JELN) of animals with different PTB-associated lesions (focal, multifocal and diffuse) and in control animals without lesions was determined by quantitative double-immunohistochemical analysis using Iba1 (ionized calcium-binding adapter molecule-1) and CK as specific markers of macrophages and epithelial cells, respectively.\u003c/p\u003e\n\u003cp\u003eAnimals with multifocal lesions showed the highest numbers of double-Iba1/CK positive cells (epithelioid macrophages (EMs)) showing significant differences with focal, diffuse and control animals in JELN and higher numbers of single-CK expressing cells in JELN and DJE. Two distribution patterns of the EMs in the granulomas were observed. In focal and multifocal animals EMs were surrounding the granuloma forming a barrier crucial to control Map infection while in animals with diffuse lesions (with significantly higher infection scores) EMs were throughout all the extension of the granuloma. Multifocal animals might be resilient to the disease as they control the shift from subclinical to the clinical through formation of ordered granulomas where EMs have a relevant role preventing Map dissemination and maintaining tissue integrity. \u0026nbsp;Since CK expression was enriched in cattle with multifocal lesions, it could be considered as a potential biomarker of PTB resilience.\u003c/p\u003e","manuscriptTitle":"Cytokeratin expression and distribution pattern of epithelioid macrophages in granulomatous lesions of animals with different pathological forms of bovine paratuberculosis: cytokeratin as a biomarker of resilience.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-10 09:04:34","doi":"10.21203/rs.3.rs-3982091/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dfcfe78a-512d-4794-be6e-16b4e1d2ec0a","owner":[],"postedDate":"May 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-25T11:01:38+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-10 09:04:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3982091","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3982091","identity":"rs-3982091","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}