Abstract
Natural killer (NK) cells play a key role in defense against Salmonella infections during the early phase of infection. Our previous work showed that the excretory/secretory products of Ascaris suum repressed NK activity in vitro. Here, we asked if NK cell functionality was influenced in domestic pigs during coinfection with Ascaris and Salmonella enterica serotype Typhimurium. Ascaris coinfection completely abolished the IL-12 and IL-18 driven elevation of IFN-γ production seen in CD16 + CD8α + perforin + NK cells of Salmonella single-infected pigs. Furthermore, Ascaris coinfection prohibited the Salmonella-driven rise in NK perforin levels and CD107a surface expression. In line with impaired effector functions, NK cells from Ascaris-single and coinfected pigs displayed elevated expression of the inhibitory KLRA1 and NKG2A receptors genes, contrasting with the higher expression of the activating NKp46 and NKp30 receptors in NK cells during Salmonella single infection. These differences were accompanied by the highly significant upregulation of T-bet protein expression in NK cells from Ascaris-single and Ascaris/Salmonella coinfected pigs. Together, our data strongly indicate a profound repression of NK functionality by an Ascaris infection which may hinder infected individuals from adequately responding to a concurrent bacterial infection.
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Introduction
Close to a quarter of the global human population is infected with Ascaris lumbricoides, a soil-transmitted helminth1,2. Young children are especially vulnerable to high-intensity Ascaris infection and chronic ascariasis which is linked to malnutrition and impaired physical and cognitive development3,4. A closely related species, Ascaris suum, is widely spread in pig farming and is frequently associated with economic losses through liver condemnation in slaughterhouses and through poor feed conversion ratio5,6. As a result of its widespread distribution, ascariasis is associated with a myriad of microbial co-infections such as Salmonella enterica both in humans and pigs7.
Infection with the facultative intracellular Salmonella enterica serovar Typhimurium is one of the most common foodborne diseases in humans8. Domestic pigs primarily act as asymptomatic carriers and natural hosts for both zoonotic Salmonella enterica and Ascaris suum infections. In addition, domestic pigs display similar physiology and a closely related organization and composition of the immune cell repertoire compared to humans9. Hence, the domestic pig provides a valuable translational animal model for studying immune responses to Ascaris and Salmonella-single infections as well as coinfections.
Ascaris-associated immune responses are marked by a type 2 (Th2) immune response characterized by elevated levels of interleukin 4 (IL-4), IL-5, and IL-13, whereby higher Th2 cytokine production predicts lower reinfection rates in humans10. In addition, Ascaris induces an immunomodulatory environment through the induction of regulatory T cells (Treg) which are thought to partake in the elevated production of IL-10 and transforming growth factor-β (TGF-β) thereby promoting host tolerance. Together with the induction of alternatively activated macrophages, the type 2/immunomodulatory environment is geared towards the limitation of adult worm burdens and promoting wound healing, the latter being essential during larval tissue migration through the liver and lungs7,11. This profile is in sharp contrast to the immune responses induced in Salmonella infection where microbial control is highly dependent on type 1 (Th1) immunity, mainly marked by the production of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α) and IL-1212,13.
In accordance with the cross-regulation between opposing programs of effector cell differentiation and function, earlier work in mice clearly showed that nematode-induced Th2 responses resulted in the impaired Th1-dependent control of coinfections with Salmonella and with other enteric microbes14. It is therefore conceivable that the immune environment induced by Ascaris infection similarly antagonizes the host's protective cellular immune responses against Salmonella infection. To date, however, it is not clear if Ascaris infection entails a higher risk for systemic salmonellosis in pigs and humans. Similarly, studies investigating the potential impact of Ascaris coinfection on the zoonotic potential of pig meat products are largely lacking. We therefore recently investigated the consequences of Ascaris coinfection for the control of acute salmonella infection and found that coinfected pigs displayed significantly higher live Salmonella burdens15.
Natural killer (NK) cells play a central role in immunity to Salmonella infections through the production of pro-inflammatory cytokines such as IFN-γ and TNF-α as well as by the release of granzyme B and perforin cytotoxic effector molecules leading to the killing of the infected host cells16,17. Porcine NK cells general activity was shown to be diminished in the presence of IL-4 and IL-2 in vitro18. Furthermore, we have previously demonstrated the suppression of IFN-γ production by NK cells following in vitro stimulation with IL-12 and IL-18 in the presence of adult Ascaris suum excretory-secretory products19. In the current study, we therefore aimed to further explore the impact of an Ascaris suum infection on NK cell functionality in an Ascaris and Salmonella coinfection setting in domestic pigs. We show that the phenotype and function of porcine NK cells is systemically altered within two weeks of Ascaris-single infection and Salmonella coinfection, evident in diminished IL-12/IL-18-induced IFN-γ production, lower cellular degranulation capacity, and the upregulation of T-bet and inhibitory NK cell receptors.
Results
Salmonella-single infection is associated with a reduction in systemic NK cell frequencies
To evaluate the impact of A. suum on NK cells during Ascaris-single infection and Salmonella coinfection, we used the domestic pig as a human-relevant model9. At day 7 post-Ascaris infection, by which time the 3rd and 4th larval stage of Ascaris undergoes hepatotracheal migration, the pigs were orally coinfected with Salmonella enterica serovar Typhimurium (Fig. 1a). Since antibodies against pan-NK markers are lacking for the pig, we adapted the gating strategy published by Gerner20 and Mair et al.21 and defined NK cells in PBMC and spleen cell lymphocytes as FSC/SSClow CD3-CD16 + CD8α + cells (Fig. 1b & Supplementary Fig. 1). The gated population homogenously expressed perforin, a hallmark of NK cells22,23 (Supplementary Fig. 1). Notably, NK cell frequencies were significantly lower (p < 0.01) systemically in both blood and spleen of the Salmonella single-infected group compared to uninfected controls, the latter displaying the expected frequencies of NK cells of healthy pigs21. In contrast, NK cell frequencies were unaffected in Ascaris-single infection compared to the uninfected control. Interestingly, Ascaris-Salmonella coinfection appeared to counteract the Salmonella-associated decline in systemic NK cell frequencies (Fig. 1b,c). Therefore, we asked whether Ascaris-single and Ascaris–Salmonella coinfection might be associated with qualitative rather than quantitative changes in NK cells and further assessed NK cell functionality upon ex vivo stimulation with cytokines.
Impaired cytokine production by NK cells during Ascaris-single and Ascaris-Salmonella coinfection
Synergistic effects of IL-12 and IL-18 are required for NK cell activation to facilitate inflammation and pathogen elimination16,24. A cardinal function of NK cells is the release of copious amounts of IFN-γ in response to IL-12 and IL-18 receptor signaling, which facilitates the differentiation/expansion of Th1 cells next to promoting phagocyte effector functions25. To assess the potential impact of Ascaris infection on NK cell effector functions, we stimulated PBMC and splenocytes with IL-12/IL-18 and quantified the ensuing IFN-γ and TNF-α responses by NK cells via flow cytometry. The CD8αdim NK cells population in both blood and spleen were the prominent IFN-γ producers (Fig. 2a). In sharp contrast to the lower NK cell frequencies seen in blood and spleen (Fig. 1), NK cells in the Salmonella-group showed a striking ability to produce IFN-γ (Fig. 2a,b) (p < 0.0001). Contrasting the unchanged NK cell frequencies, NK cells present in the blood and spleen of Ascaris-single infected pigs displayed significantly impaired IFN-γ responses to IL-12/18 stimulation (p < 0.01) (Fig. 2a,b). A similar effect was seen comparing the Ascaris–Salmonella coinfected group to Salmonella-single infected pigs, whereby NK cells of the latter displayed the most vigorous IFN-γ expression upon cytokine stimulation. Though in limited amounts, TNF-α production by NK cells was only evident in Salmonella-single infection both in blood and spleen (Supplementary Fig. 2a). Taken together, these results show that Ascaris suppresses IFN-γ production by NK cells during single infection, but also in pigs concurrently infected with Ascaris and Salmonella in response to IL-12 and IL-18 stimulation.
Ascaris impairs the cytolytic/degranulation potential of NK cells in Ascaris–Salmonella coinfection
IL-12 and IL-18 produced by Salmonella-infected macrophages and dendritic cells are essential prerequisites to trigger NK cell degranulation16. The expression of the lysosomal protein CD107a (Lysosome-Associated Membrane Protein 1, LAMP-1) is correlated with NK cell cytotoxicity26. Therefore, we evaluated NK cells surface expression of CD107a following stimulation with IL-12 and IL-18 cytokines. Interestingly, Ascaris significantly suppressed the expression of the degranulation marker CD107a compared to the controls (p < 0.01) both in blood and spleen. The NK cell degranulation repression was also evident in the coinfected group compared to the controls (p < 0.01) and Salmonella-single infected group (p < 0.0001) (Fig. 3a,b).
IL-18 can also prime NK cells’ cytotoxicity thus enhancing the release of perforin which forms pores on the infected cells facilitating the uptake of granzymes which induces cell lysis or apoptotic death27,28. In agreement with the higher expression of the degranulation marker CD107a, perforin was also upregulated in the Salmonella-single infected group (Fig. 3c). This increase in perforin expression was inhibited in the Ascaris-Salmonella coinfection setting (Fig. 3c,d). In contrast, the Ascaris-single infected group differed from the Ascaris-Salmonella coinfected group as there was a significant upregulation of perforin in the Ascaris-group compared to the controls. The higher CD107a expression by the splenic NK cells of the Salmonella single-infected group was associated with significantly higher levels of CD8α expression (p < 0.001) compared to the coinfected group (Supplementary Fig. 2b). This upregulation of CD8α was not evident in the peripheral blood NK cells. In addition, blood and splenic NK cells of the Salmonella-single infected pigs showed a two-fold increase in the percentages of the CD107a + NK cells compared to Ascaris-single and Ascaris-Salmonella groups (Supplementary Fig. 3a,b). These results suggest that NK cell cytotoxicity is inhibited during an acute Ascaris infection and in coinfection with Ascaris and Salmonella enteric microbes. Notably, systemic NK cell numbers are low in Salmonella single-infected pigs (Fig. 1), but the NK population is enriched in cells displaying high responsiveness to cytokine signals as well as elevated expression of degranulation markers. The decline in systemic NK cell frequencies might be partially explained by the extensive expression of NK effector functions and subsequent cell death in Salmonella single-infected pigs, as we determined moderately higher rates of NK cells marked by viability dyes in flow cytometry analyses of samples from this group.
Reduced functionality of NK cells in Ascaris–Salmonella coinfection is associated with upregulation of T-bet transcription factor
We next investigated the NK cell expression of the transcription factors Eomesodermin (EOMES) and T-bet which are the master regulators of NK cell maturation, differentiation and functionality29. T-bet is key for NK cells’ terminal differentiation and induction of the inhibitory receptors30. EOMES is mainly expressed by immature NK cells though they balance their expression with T-bet to facilitate NK cells maturation. EOMES-deficient NK cells exhibit poor cytotoxicity, whereas T-bet deficient NK cells possess a normal cytotoxicity functionality31. We therefore evaluated the associations between the balance of these transcription factors and the functionality differences observed above. Strikingly, NK cells from Ascaris-infected pigs were marked by the highly significant upregulation of T-bet protein expression levels (p < 0.0001) compared with the uninfected controls (Fig. 4a,b). In line with the functional differences observed above, significant upregulation of T-bet transcription factors (p < 0.0001) in the Ascaris-Salmonella group compared to the control and Salmonella-single groups was noticed. Conversely, there were no significant changes in EOMES expression between the groups (Fig. 4c). However, EOMES-positive NK cells were prominent in IFN-γ production (Fig. 4d). Together, these data indicate that Ascaris modulates porcine NK cells through upregulation of T-bet possibly interfering with NK cell maturation and terminal differentiation.
Ascaris modulation of NK cells is associated with upregulation of natural killer inhibitory receptors
The activation and regulation of NK cell function depends largely on the activating and inhibitory receptors32. Considering that T-bet overexpression is associated with terminal differentiation and upregulation of inhibitory receptors, we determined gene expression levels of inhibitory receptors (KLRA1/Ly49 and NKG2A) which facilitates MHCI interactions and NK functionality30. In addition, we evaluated gene expression of the natural cytotoxicity receptors (NCR) (NKp46 and NKp30) which are readily expressed upon EOMES overexpression30. Based on the mRNA profiles determined with FACS-sorted splenic NK cells from the different groups, we found that NK cells from Ascaris-single infected and Ascaris–Salmonella coinfected pigs were marked by the significantly upregulated expression of NK cell inhibitory receptors compared to uninfected controls and Salmonella-single infected groups (Fig. 5a,b). There was no upregulation of NCR genes in the Ascaris group though we noticed minimal but significant upregulation of NKp46 and NKp30 in the Ascaris-Salmonella coinfected group (p = 0.01) (Fig. 5c,d). The upregulation of NCRs in the coinfection group without the matching increase in functionality may be attributed to the counter effects of NK cell inhibitory receptors overexpression by Ascaris infection. In addition, we determined CXCR3 chemokine gene expression on the sorted splenic NK cells since expression of CXCR3 is associated with T-bet expression and cell trafficking in murine models33. Significantly higher expression of CXCR3 genes (p = 0.03) was observed in the Ascaris group compared to the controls indicating better homing capacity and possible higher interaction with T-bet. Overall, our results therefore suggest that Ascaris hinders the effector functionality of NK cells through the upregulation of NK cell inhibitory receptors.
Discussion
Ascaris infections occur predominantly in developing countries which often lack adequate sanitation thereby bearing a higher risk of coinfection with other microbial pathogens such as Salmonella34,35. As a major cause of foodborne diseases in humans, Salmonella infections pose a huge public health problem globally with chicken and pigs representing the main contaminated meat source36. The emergence of infections with multi-drug resistant Salmonella in addition to the lack of an optimal vaccine calls for a better understanding of the host immunity37. This gains further relevance in the context of helminth coinfections which can antagonize the host's protective responses against Salmonella and other intracellular pathogens7. Domestic pigs not only serve as an important meat source but also as important carriers of zoonotic A. suum and Salmonella infections6,9,38. The domestic pig thus offers a valuable model for translational research as part of a one health approach.
The severity of Salmonella infections is dependent on the infecting serovar, the infection dose, age, and the immunological status of the host39. The subspecies Salmonella enterica serovar Typhimurium is a major cause of clinical diseases both in humans and animals. To date, little is known about the role of NK cells in helminth and bacterial co-infections both in humans and pigs. In the murine model, NK cells plays a prominent role in the control of Salmonella infections during the early stages16,40,41. Furthermore, helminth-salmonella coinfection in mice leads to impaired host resistance against Salmonella14. We have previously demonstrated in vitro modulation of NK cells and dendritic cells by Ascaris excretory-secretory products19. Here we aimed to gain more insight into the possible immunomodulatory effects of Ascaris on NK cells during coinfection with Salmonella in domestic pigs, which are their natural host.
Our results show that acute Ascaris infections significantly compromise the capacity of NK cells to produce IFN-γ in pigs. This Ascaris-induced modulatory effect was similarly evident in the Ascaris–Salmonella coinfection group. In support of our findings, both in murine models and in vitro experiments, NK cells rapidly produce IFN-γ in response to Salmonella infection16,17,42. In addition, among other immune cells, NK cells are the largest population of IFN-γ producers in response to IL-12 and IL-18 stimulation16. Following the breaching of the epithelial barrier, Salmonella preferentially invades gut resident macrophages which respond by activating the neighboring NK cells both by direct cell-to-cell contact and through the release of IL-12 and IL-18 cytokines43. Activated NK cells in turn produce IFN-γ which further activates the infected macrophages16. IFN-γ enhances the release of reactive oxygen and nitrogen species, hydrolytic enzymes and antimicrobial peptides leading to phagolysosome enrichment and subsequent killing of the intracellular bacteria44. Deficiencies in the IL-12/IFN-γ axis in mice and humans increase susceptibility to Salmonella infections through impaired granuloma formation, resulting in uncontrolled bacteria growth inside the macrophages45,46. Thus, inadequate IFN-γ production in Salmonella infections as a result of Ascaris infection as seen in the current study would potentially antagonize the protective host immunity against Salmonella. In concordance with the Ascaris-induced impairment of NK cell functionality, the coinfected pigs had higher live Salmonella burdens compared to the Salmonella-single-infected group15, though the mechanistic link with NK cells functionality calls for further investigation.
We show that Ascaris potentially suppresses the cytotoxic function of NK cells as exhibited by the significantly lower surface expression of the degranulation marker CD107a (LAMP-1). Furthermore, the NK cells' degranulation potential was impaired in the coinfection setting which was coupled with strongly reduced levels of perforin compared to the Salmonella-single group. In mice, IL-18-mediated NK cellular cytotoxicity through perforin responses is crucial for resistance against salmonellosis during the early stages of infection28. The hyporesponsiveness to IL-12 and IL-18 stimulation for both cytotoxicity responses and IFN-γ production observed in the Ascaris-single infection and coinfection implies that Ascaris clearly impedes the functionality of NK cells.
Whether NK cells exert direct immunological control over the Ascaris larvae remains unkown. Of note, the depletion of murine NK cells early during infection with the gastrointestinal nematode Heligmosomoides polygyrus infection did not affect the worm burden but rather resulted in increased endothelial injury47. This may suggest that Ascaris also modulates the effector functions of NK cells in its natural hosts either to avoid larval killing, possibly through the repression of NK cell cytotoxic functions, or to benefit from NK activity associated tissue homeostasis and wound healing. However, this complex interaction remains to be elucidated. The chemokine receptor CXCR3 (CD183) is associated with T-bet and the recruitment and trafficking of murine immune cells33. Thus, the upregulation of CXCR3 genes to possibly increase NK cell trafficking capability by acute Ascaris infection without the induction of cytokine production and cytotoxicity function suggests that NK cells are still required for other functions such as immune regulation and/or wound healing to promote disease tolerance.
We show that Ascaris modulates the functionality of NK cells by upregulating the expression of the T-bet transcription factor. Murine NK cell development, maturation, and functionality is orchestrated by the balances between EOMES and T-bet transcription factors31. Whilst EOMES is dominantly expressed in immature NK cells, T-bet is specifically essential for the terminal differentiation of NK cells and epigenetically promotes the expression of NK cell inhibitory receptors expression30. This is in agreement with the findings of this study whereby the induction of terminally differentiated NK cells by Ascaris was significantly associated with (Ly49/KLRA1, and NKG2A/KLRD1) inhibitory receptors gene expression. Thus, Ascaris-induced impairment of NK cells may be attributed to the upregulation of NK cell inhibitory receptors which are key checkpoints inhibitors controlling the activation of NK cells.
The natural cytotoxicity receptors NKp46 (CD335), NKp44 (CD336), and NKp30 (CD337) are essential for NK cell degranulation and cytokine production32. Although the majority of porcine NK cells are NKp46 negative, CD8αdimNKp46high NK cells are highly activated possessing a higher IFN-γ producing capacity as well as the enhanced ability to release cytolytic granules following IL-12 and IL-18 stimulation21,48. Accordingly, CD8αdim NK cells showed the highest IFN-γ production capacity in the current study. Thus, the downregulation of the activating receptor genes by Ascaris together with the upregulation of inhibitory receptor genes would hinder both the cytokine and degranulation capacity of the NK cells as observed here. This study was limited to assess NKp46 expression in NK cells.
In conclusion, our data show that NK cell functionality is impeded by acute Ascaris infection in pigs. This Ascaris-associated impairment of NK cells was also evident in the Ascaris-Salmonella coinfection setting. Further elucidation of the effects of the acute and also chronic Ascaris infections on NK cells in promoting susceptibility, bacterial burdens and severity of Salmonella infection in pigs and/or in humans will be of uttermost importance. In addition, with the evolution of memory in NK cells49, evaluation of the longevity and mechanisms of the NK cell reprogramming by Ascaris would be essential as NK cells are crucial for intracellular pathogen and tumor control.
Methods
Ethics statement
Ethical approval was obtained from the State Office of Health and Social Affairs Berlin, Germany (Landesamt für Gesundheit und Soziales Berlin, Germany) (G0212/20). The study was performed following the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes and the German Animal Welfare Law. This study is reported in accordance with ARRIVE guidelines 2.0.
Ascaris suum and Salmonella enteritica infection
24 German landrace hybrid pigs (12 males and 12 females) from a conventional breeding facility (Brandenburg district, Germany) at 6–7 weeks of age, were used for the experimental infections. A"trickle-like" Ascaris infection regimen was adopted from previously reported doses (< 10,000 eggs) to mimick a ‘natural’ exposure setting50. A sub-clinical dose was used for Salmonella infections51. 6 pigs (Ascaris group) were orally infected with 2000 embryonated Ascaris suum eggs on 4 consecutive days (total inoculation dose of 8000 eggs, embryonation rate 97.80%). Ascaris suum egg collection, density purification and evaluation of the embryonation rate were carried out as previously reported52. The Ascaris group pigs were dissected two weeks post-infection. The Salmonella group entailed 6 pigs that were orally infected with Salmonella Typhimurium definitive type 104 with a single inoculum dose of 107 colony-forming units (CFU) and dissected one week post-infection. Similarly, the Ascaris-Salmonella coinfection group (n = 6) was orally infected with 2000 embryonated Ascaris suum eggs on 4 consecutive days (total inoculation dose of 8000 eggs), and co-infected with Salmonella enterica serovar Typhimurium (inoculum dose of approximately 107 CFU) one week after Ascaris infection followed by dissection one week later. The pigs were kept for 14 days to allow them to acclimatize before the experimental infections. During this period, the pigs were also thoroughly examined for any pre-existing helminth or Salmonella infections. All pig groups, including the uninfected group (n = 6), were kept in separate indoor pens with a controlled light system. In addition, the stables were equipped with a centrally controlled heating system. The pigs received a standard diet based on their body weight, which was determined once a week and water was given ad libitum.
Blood collection and isolation of porcine peripheral blood mononuclear cells
Blood was collected in EDTA tubes (S-Monovette, Sarstedt) by heart puncture after sedation with ketamine hydrochloride (20 mg/kg BW, Ursotamin, Serumwerk Bernburg AG, and 2 mg/kg BW), azaparone (Stresnil, Janssen-Cilag GmbH, Germany) and xylazine (36 mg/kg BW; Xylavet; CP-Pharma Handels GmbH). Peripheral blood mononuclear cells (PBMC) were isolated from whole blood by density gradient centrifugation using 0.9% sodium chloride solution at a 1:1 ratio using pancoll (1.077 g/ml density; PAN-Biotech GmbH; Aidenbach, Germany) using Leucosep tubes (Greiner Bio-One, Austria). Following the density gradient centrifugation at 1500 rpm for 20 min at room temperature (RT), PBMC were carefully obtained at the interface and washed in 0.9% sodium chloride. The remaining erythrocytes were lysed using erythrocyte lysis solution (H2O plus 0.01 M KHCO3, 0.155 M NH4Cl, and 0.1 M EDTA, pH 7.5) at RT for 5 min. PBMC were thereafter washed twice and resuspended in complete Iscove’s modified Dulbecco’s medium (cIMDM; IMDM with stable glutamine, 25 nM HEPES and 3.024 g/l NaHCO3, plus 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin; PAN-Biotech GmbH; Aidenbach, Germany).
Isolation of splenocytes
Pooled tissue sections from the spleen were sampled following euthanization of the pigs by intracardial injection of tetracaine hydrochloride, mebezoniom iodide, and embutramide (10 mg/kg BW; T61, Intervet, Germany). The spleen sections were mechanically disrupted and the suspension passed through a 70 µm cell strainer. Washing, erythrocyte lysis and pellet resuspension were carried out as described for the PBMC isolation.
Stimulation of NK cells with IL-12 and IL-18
Freshly isolated PBMC and splenocytes were counted using the CASY automated cell counter and cultivated in cIMDM at a density of 1 × 106 cells/ml in a final volume of 200 μl. Cells were stimulated with either 25 ng/ml porcine IL-12p70 and 100 ng/ml porcine IL-18 (R&D Systems; Minneapolis, USA) or left in the culture medium alone as negative control and incubated overnight at 37 °C and 5% CO2. Brefeldin A (ThermoFisher; Waltham, MA, USA) was added after 2 h of stimulation at a final concentration of 3 μg/ml. The NK cells degranulation capacity was assessed through the surface expression of CD107a following IL12/18 stimulation. During the addition of Brefeldin A, the cultures were supplemented with Alexa Flour® 647 conjugated mouse anti-pig CD107a antibody (IgG1, clone 4E9/11, Bio-Rad).
Flow cytometry analysis
Before flow cytometry (FCM) staining for the analysis of NK cells both the stimulated and unstimulated PBMC and spleen cells were resuspended in PBS (PAN-Biotech GmbH; Aidenbach, BY, Germany) containing 5% (v/v) FCS and 2 mM EDTA. Mouse serum (1:500) was used for blocking prior to staining. Surface markers (CD3, CD8α and CD16), cytokines (IFN-γ and TNF-α), transcription factors (EOMES and T-bet) and perforin were stained using pig-specific or cross-reactive antibodies outlined in (Supplementary Table 1). The fixable viability dye eFluor® 506 (ThermoFisher; Waltham, MA, USA) was used for dead cell exclusion. Cells were fixed and permeabilized with a Transcription Factor Staining Buffer Set (eBiosciences) to facilitate intranuclear staining. All incubation steps were performed for 10 min at 4 °C. The FCM analysis of NK cells was performed on BD FACSAriaIII (BD Biosciences). FCM data was acquired using the FACSDiva software (BD Biosciences) and evaluated using FlowJo software (version 10.0, Tree Star, Ashland, OR, USA).
FACS sorting of CD3-CD8α + CD16 + NK cells from spleen cells was performed on BD FACSAriaIII (BD Biosciences). In addition, eFluor® 506 fixability dye was included to discriminate between the live and dead cells. The average purity of the sorted NK cells was 95.0%. The NK cells were directly sorted in RNAlater® (Sigma-Adrich, Germany) and stored at − 80 °C for further gene expression analysis.
Evaluation of gene expression by quantitative reverse transcriptase PCR
Total RNA from the sorted NK cells was extracted using the RNeasy Mini kit (Qiagen, Germany) following the manufacturer’s protocol. The amount and the purity of the extracted RNA was determined using a Nanodrop ND2000 spectrophotometer (Nanodrop Technologies) at 260/280 nm. Gene expression from the unstimulated NK cells was evaluated using the Light-Cycler® 480 II system (Roche) following total RNA transcription into cDNA using the High Capacity RNA-to-cDNA kit (Applied Biosystems). SYBR® Green I Master Mix (Roche) was used for the amplification of the target genes. Internal standard from equal pooled cDNA of all the samples analysed in this study was used for standard curve generation for subsequent gene expression evaluations. Primers used for all gene targets were adopted from48,53, and all primers were commercially synthesised (TIB-Molbiol, Germany). Sequences for all primers are provided in (Supplementary Table 2). To normalize target-gene expression the housekeeping genes of ribosomal protein L19 (RPL19) and glycerinaldehyd-3-phosphat-dehydrogenase (GAPDH) were used. Samples were analysed in duplicate. Relative expression of the target genes was calculated using the efficiency-corrected 2−ΔΔCT method54.
Statistical analysis
Statistical analysis was done using GraphPad PRISM version 9.0 (GraphPad Software, USA). Normality was tested with the Shapiro–Wilk test. One-way variance analysis (ANOVA) with Bonferroni correction for paired sample means was applied, p < 0.05 was considered significant.
Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.
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Acknowledgements
We are grateful to Marion Müller, Yvonne Weber, Bettina Sonnenburg, Christiane Palissa, and Beate Anders all from the Institute of Immunology, Freie Universität Berlin for their immense support, efforts in sample processing and technical support. We thank Dr. Anne Winkler (Institute of Immunology, Freie Universität Berlin) for designing the graphical illustrations. We also acknowledge Benjamin M. Hamid (Current affiliation: University of Veterinary Medicine, Vienna, Austria) for his technical assistance. Special thanks to all the animal keepers at Freie Universität Berlin Department of Veterinary Medicine.
Funding
Open Access funding enabled and organized by Projekt DEAL. This work was funded by the Deutsche Forschungsgemeinschaft (DFG) Grant HA 2542/11-1 to SH and HA 2544/1-1 to SR and a grant from the Einstein Foundation Berlin to SH. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Conceptualization: SH, SR, RMM & AM. Data analysis: SR, RMM & LO. Funding acquisition: SH. Investigation: RMM, LO, AM, SR, JSB, AL, JA, AK, PH, ZDM & RH. Supervision: SH, SR, AM & JBS. Writing—original draft: RMM & LO. Writing—review and editing: RMM, LO, AM, ZDM, JA, AK, AL, RH, PH, JSB, SR & SH.
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Mugo, R.M., Oser, L., Midha, A. et al. Acute Ascaris infection impairs the effector functions of natural killer cells in single and Salmonella co-infected pigs. Sci Rep 14, 14586 (2024). https://doi.org/10.1038/s41598-024-64497-4
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DOI: https://doi.org/10.1038/s41598-024-64497-4
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