Effect of Vitamin D3 On the Expression of Antimicrobial Peptide Gene in Experimental Pneumonia in Calf


 Background

Vitamin D3 has been identified as an immunomodulatory agent that confronts the pathogens via stimulating antimicrobial peptides (AMPs).
Objective

The effects of vitamin D3 on the expression of AMPs was assessed in experimental pasteurellosis in calves.
Methods

10 Holstein crossbred male calves (2–4 months) were chosen and randomly divided into the two groups. Pasteurella multocida was prepared (3×109 CFU/mL) and inoculated in the trachea. Vitamin D3 was injected to the treatment group after confirming the pneumonia. Blood samples were obtained from both groups at different time intervals and the peripheral blood mononuclear cells (PBMCs) were isolated. Clinical symptoms were recorded. Broncho-alveolar lavage was performed to evaluate the lung cell content. On the other hand, 10− 6, 10− 7, and 10 − 8 molar (M) of vitamin D3, was used to evaluate the expression of CD4, BMAP34, and BNBD4 genes using PBMCs under the in vitro conditions.
Results

The prescription of vitamin D3 to the treatment group caused a decline in clinical signs. Following the vitamin D3 injections the treatment groups under the in vivo conditions, significant increase was observed in the expression level of Defensin (BNBD4), and CD4. Evaluation of bronchoalveolar lavage fluid (BALF) revealed that the amount of neutrophils decreased after vitamin D injection. In vitro, increased expression of Catalicidin (BMAP34), Defensin (BNBD4), and CD4 was observed at a concentration of 10− 6 M of vitamin D3.
Conclusion

The present study indicated that vitamin D3, exerts immunomodulatory effects on many infectious diseases via activation of VDR pathways and stimulation of AMP production.


Introduction
Calf pneumonia is a major problem in dairy and beef herds and can be caused by different etiologies; therefore it is termed a 'multifactorial disease'. Various factors including viruses (respiratory syncytial virus (RSV), bovine viral diarrhea (BVD), infectious bovine rhinotracheitis (IBR)), bacteria (Pasteurella multocida, Mannheimia haemolytica), inadequate colostrum intake, poor nutritional status and hygiene in pens, insu cient ventilation, over crowdedness, and stress lead to pneumonia (Dabo et al. 2008;Smith 2015).
The outbreak of cattle pneumonia in different regions of the world has been reported between 10-70% (Gibbs 2001; Mohammadi et al. 2006). The mortality rate due to respiratory diseases in dairy cattle has also been reported between 2.2-9.4%. Approximately 3% of born calves have been died due to pneumonia in the rst 12 weeks of their lives (Geraert 2006; Mohammadi et al. 2007; Constable et al. 2017).
Pasteurellosis is one of the most important contagious diseases in the world. Pasteurella multocida and Mannheimia haemolytica are considered as the commensal gram-negative bacteria in the upper respiratory tract of animals. Under stresses including environmental, managemental, or infectious factors, those agents can cause growth and extension of bacteria to the lower respiratory tract and often produce mild to severe clinical signs (Smith 2015; Mohammadi et al. 2007; Selvaraj et al. 2009; Karimkhani et al. 2011).
The clinical manifestations include a rise in temperature, respiratory distress with nasal discharge, and then in severe cases, recumbence and death may be the result (Smith 2015;Constable et al. 2017).
Vitamin D 3 not only plays a signi cant role in calcium homeostasis and bone metabolism, but also modulates innate and adaptive immunity in the respiratory and cardiovascular systems (Bikle 2009; Guillot et al. 2010).
Recently, the role of vitamin D 3 signaling in the modulation of innate immunity has been indicated. As immune system stimulation due to bacterial infection can cause change 25D into 1, 25 D. Furthermore, produced 1,25D induces the expression of genes encoding antimicrobial peptides (AMPs), speci cally Cathelicidin antimicrobial peptide (CAMP) (White 2010). An e cient immune system requires adequate vitamin D 3 levels for the expression of AMPs in some epithelial cells (Anderson and Rings 2008). Moreover, vitamin D 3 exert broad anti-in ammatory effects on the adaptive immune system and can increase the number and/or function of T regulatory cells (Tregs).
Researchers have highlighted the correlation of low levels of vitamin D 3 with an increased risk of infections of AMPs are an important part of the intrinsic and acquired immunity of most living organisms against pathogens. AMPs are small molecular weight proteins which reveal broad-spectrum activity against microorganisms such as gram-positive and gram-negative bacteria, viruses, yeasts, protozoa, and fungi (Reddy et al. 2006;Beisswenger and Bals 2005). A variety of cells including epithelial and phagocytic cells such as macrophages, neutrophils, and natural killer cells synthesize and secrete AMPs. The mechanism of action of AMPs includes the involvement, damage, and rupture of the outer cell membrane of the organism (Beisswenger and Bals 2005).
The Cathelicidin family of AMPs have been isolated from several different types of mammals and hence are considered as important AMPs with linear α helical domains (Reddy et al. 2004). They play a signi cant role in mammalian innate immune defense against invasive bacterial and viral infections. In cattle, different Cathelicidin were named Bac5, Bac7, BMAP27, BMAP28, and BMAP34 (Zantetti 2004). Additionally, Defensins are identi ed as a member of AMP families' and are structurally divided into α, β, and θ-Defensins. In cattle, β Defensin is generally found in neutrophils, alveolar macrophages, trachea, colon, small intestine, lung and spleen this type of Defensin varies in sites of expression, structure, and biological activities (Tecle et al. 2010;Hazlett and Minhao 2011). Defensins act by disrupting the structure of bacterial cell membranes. They also play a major role in defense against pathogens in many organs. These peptides have a wide range of antimicrobial activities and are recognized as novel therapeutic agents because they enhance immune system activity as immune modulators.
Their best feature is recognized as their ability to eliminate invasive agents such as gram-negative and grampositive bacteria, fungi and viruses. However, nowadays it has been determined that these peptides are able to attract the in ammatory cells and activate adaptive and innate immune responses (Tecle et al. 2010;Hazlett and Minhao 2011).
In vitro studies showed that vitamin D 3 has therapeutic and anti-in ammatory effects via modulating the innate and adaptive immune system. So that, vitamin D 3 can be up-regulating the expression of T cells such as CD4 and AMPs.
Due to the high prevalence of respiratory diseases in human societies, the application of vitamin D 3 as an auxiliary treatment for activating the AMPs interacting with innate and adaptive immune receptors deems useful.
Therefore, the intake of vitamin D 3 in therapeutic and prophylactic doses is recommended, and in this regard, in the present study, we aimed to evaluate the immunomodulatory effect of vitamin D 3 on the mRNA expression of AMPs in experimental pneumonia in calves.

Materials And Methods
Experimental study design For this study, based on accepted criteria by Iran National Research Ethics Committee, ten Holstein male crossbred calves (2-4 months of ages) for experimental study were chosen. The calves were selected from an industrial farm of Tehran suburb and transferred to the Research Veterinary Hospital after initial clinical examinations and proved their healthy conditions by veteran. The criteria for entrance into the study were normal rectal temperature, respiratory sounds, heart rates and lack of nasal and eyes discharge. Subsequently, calves were randomly divided into the treatment and control groups by veterinary hospital staffs, numbered and adaptation period carried out about 6 days after entrance day. The calves were kept individually in a closed and isolated pens designed for large livestock, with appropriate ooring, temperature, food, and water. The experimental study sites were thoroughly isolated and investigated during the study to completely prevent the risk of any other infection. The blood samples (10 mL) were obtained from the jugular vein before the beginning of the study, at the challenge time (bacterial inoculation or 0 hours of the study), 24, 48, 72, 96, and 120 hours of study, and poured into the sterile tubes containing lithium heparin anticoagulant for the isolation of peripheral blood mononuclear cells (PBMCs). In the beginning of the study, the calves were restrained and the tracheal tube was gently inserted into the trachea and xed. Then lavage catheter was inserted and the prepared Pasteurella multocida (PMC66) was inoculated by the nal volume 10 mL (3×10 9 CFU / mL).
Isolation and preparation of the bacteria Pasteurella multocida isolated from the lung of a calf with pneumonia by Razi Vaccine and Serum Research Institute, was grown in phenol red-free RPMI 1640 medium supplemented with 200 mM L-glutamine and diluted in sterile phosphate buffered saline (PBS), pH 7.4. To con rm the species of bacteria, bacterium-speci c primers were designed by ALLELE ID software and Polymerase Chain Reaction (PCR) was performed accordingly. The toxA gene of the Pasteurella multocida was ampli ed using two standard forward and reverse primers (Table1) (Abed et al. 2020; Algammal et al. 2020). PCR was performed in a nal reaction volume of 20 μL containing 10 μL of 2× PCR Master Mix, 1μL of each primer (both from SinaClon BioScience, Iran), 2 μL template DNA and 6 μL double-distilled water. Then PCR ampli cation was performed as follows: initial denaturation at 95°C for 5 min, 35 cycles of denaturation at 95°C for 10 s, annealing at 52°C for 20 s, and extension at 72°C for 60s, and nal extension at 72°C for 5 min. Finally, PCR products were analyzed using 1% agarose gel electrophoresis.

Inoculation of bacteria
According to previous and pilot study, appropriate dilution of bacteria (3 × 10 9 CFU / mL) was prepared. Before launching study, the calves were restrained. The tracheal tube was inserted gently into the trachea and the tube cuff was xed. Then quickly lavage catheter was inserted through the endotracheal tube which inoculated the prepared Pasteurella multocida (PMC66) for experimental pneumonia induction by the nal volume of 10 mL.

Clinical signs evaluations
The clinical signs such as rectal temperature, ocular and nasal discharge, cough, abnormal breathing and auscultation of abnormal lung sounds were evaluated by the practitioner in the calves before the intervention, after inoculation, and every 24 hours to correctly classify the calf and identify required therapeutic interventions.
Calves with this infection has at least 2 clinical signs of respiratory infection, and therefore were considered as pneumonia cases (Constable et al. 2017;DeRosa et al. 2000).

Injection of Vitamin D 3
After diagnosis of calves with pneumonia by clinical examination, 10 mL of vitamin D 3 (Daropakhsh® Co.) was injected intramuscularly to the treatment group.

Total blood cell count
The blood samples were collected in sterile tubes containing the EDTA and transferred to the laboratory to be analyzed by auto cell counter. To differentiate white blood cells, smears were prepared, xated by alcohol and stained by Wright Giemsa (1: 5 ratio).

Bronchoalveolar lavage (BAL)
Bronchoalveolar lavage (BAL) was performed in the calves anesthetized with Propofol (Fresenius Kabi, U.S.A.) (Diprivan®5mg/kg) using a sterilized and exible catheter with a 3-5 cc balloon cuff (Supa Co, Iran) at the challenge time (start time (0h) of study or bacterial inoculation) , 24 h after bacterial inoculation, 48 h bacterial after inoculation, and in the end of the study (120 h). The BAL catheter was inserted into the trachea using a tracheal tube, and its positioning was con rmed by repeated coughing. Then, the balloon cuff, in ated with 3 mL of air and subsequently, 5 aliquots of 200-300 mL pre-warmed sterile saline solution (37 o C), was infused, and immediately after infusion, the lavage uid was aspirated applying negative pressure. The lavage uid was mixed and pooled in a sterile tube maintained on ice and immediately transferred to the biochemistry laboratory. The calves were under critical care after the BAL procedure to prevent any bronchial complications.
To obtain a cell-free supernatant, BALF was centrifuged at 450 g for 15 min. For cytological evaluation of BALF, the cells were precipitated by centrifugation and stained using Wright-Giemsa staining and direct smear preparation.
Blood sampling and isolation of peripheral blood mononuclear cells (PBMCs) The collected blood samples were diluted by sterile PBS (Sigma-Aldrich) added gently and slowly to Ficoll Histopaque (5 mL) (Biowest Co, Germany). The whitish buffy coat (PBMCs) which was formed in the inter-phase between histopaque and the medium was aspirated. PBMCs isolated from the whole blood were re-suspended in RPMI1640 (

Statistical analysis
Data were tested for normal distribution of the parameters with Kolmogorov-Smirnov test. The comparisons of different time intervals of the study were performed using repeated measurement ANOVA. Moreover, the results of this study were analyzed using the independent T test and post hoc Tukey test. Additionally, for the analysis of data before and after vitamin D 3 injections, we used paired t test. Differences with P values of less than 0.05 were considered statistically signi cant.

Con rmation of the isolated bacteria
According to the primers designed, PCR was performed to con rm the species of bacterium. The images obtained on the gel electrophoresis revealed the Pasteurella multocida with a molecular weight of 865 bp (Fig. 1)

Assessment of clinical signs
Clinical signs of pneumonia were monitored during the rst 12 hours after bacteria inoculation so that elevated rectal temperature, nasal and in some cases eyes discharge, coughing, and abnormal respiratory sounds were recorded in both groups. In the treatment group, after vitamin D 3 injection, the overall clinical symptoms decreased in comparison with the control group, along with a complete recovery which was observed in some calves after 120 hours (Fig. 2).

Hematological and Biochemical appearance
After bacterial inoculation, signi cant increases were observed in total white blood cell (WBC), total proteins, lymphocytes, and eosinophils within 24 hours of the study. However, WBC count, total protein, lymphocyte, and eosinophil count decreased after the administration of vitamin D 3 in the treatment group in comparison with the control group. These ndings, with clinical signs, indicated an improvement in the symptoms of pneumonia in the treatment group compared to the control group (P < 0.05).
According to the hematological ndings, no signi cant differences were observed between the two groups in terms of blood components (P>0.05), which con rmed the calves' health conditions before inoculation of bacteria.
Fibrinogen levels in two groups increased after bacterial inoculation and in the control group remained in high level compared to the calves received vitamin D 3 (P < 0.05).

Evaluation of bronchoalveolar lavage uids
There was a signi cant difference between the control and treatment groups in terms of cell contents evaluated in the BAL (P=0.0001). The number of BALF neutrophils at the challenge time and on the last day of the study (120 hours) were signi cantly different in comparison to their number before challenge (P=0.0001) (Fig. 3). Furthermore, quantitative analysis of mast cell counts at different hours showed that the number of cells at the time of vitamin D 3 injection was signi cantly different from their number at other study hours (P=0.0001) (Fig. 4).
Additionally, analysis of BALF lymphocytes at different hours of the study showed that the number of cells at challenge time was signi cantly different from their content at the time of vitamin D 3 injection and at 48 hours of the study (P=0.0001) (Fig. 5). Moreover, the evaluation of BALF macrophages revealed that the number of cells were signi cantly different at 24 and 72 hours of study in comparison to that at other time points (P=0.0001) (Fig.   6). However, the eosinophil numbers revealed no signi cant differences between the two mentioned groups.

Gene expression of antimicrobial peptides in infected animals
Signi cant differences were observed between the control and treatment groups (P = 0.04) in terms of the level of De nsin however differences were not signi cant considering the time points (Fig. 7).
BMAP34 gene was considered for the expression of the Cathalicidine AMP. No signi cant changes were identi ed between the control and treatment groups in the BMAP34 gene expression at different hours of the study.
Nonetheless, a signi cant difference was observed in the CD4 gene expression between the control and treatment groups (P=0.0001) to such a degree that CD4 gene expression changes between the control and treatment groups at 48, 72, and 120 hours after the challenge time were signi cantly different ( P <0.05) (Fig. 8).

Gene expression of antimicrobial peptides in isolated cells in vitro condition
In vitro evaluation was performed to compare the results with the in vivo conditions. The concentration of vitamin D 3 applied in this study were 10 -6 , 10 -7 , and 10 - Hence, supplementation with 1, 25 dihydroxycholecalciferol increased the expression of Cathelicidin, and restricted the replication of Mycobacterium tuberculosis (Mtb) in human macrophages (Afsal et al. 2014).
An experimental study in sheep showed that the bacterial load decreased in the pulmonary tissues and BALF following the injection of 0.5 mg Cathelicidin (Izadpanah et al. 2005). In the present study, Vitamin D 3 could increase the in vivo expression of Defensin and CD4 with a dose of 3000000 IU, as well as increasing the expression of Defensin, Cathelicidin, and CD4 in cell culture with a higher dose.
Administration of different doses of vitamin D 3 may improve the immune system function, and decrease the severity of bacterial and viral infections in livestock. On the other hand, the vitamin D 3 de ciency may act as a risk factor in the dysfunction of defensive system especially respiratory organs.
Some blood parameters such as white blood cells, lymphocyte, eosinophil, and brinogen increased after the inoculation of bacteria.
The results obtained from BALF assessment showed that the level of neutrophils increased following the experimental pneumonia, while it decreased after vitamin D 3 injection. The macrophage contents also increased after vitamin D 3 injection in the treatment group. The cell contents and BALF cell patterns for the expressions of antimicrobial genes con rmed the immunomodulatory and antimicrobial effects of vitamin D 3 in experimental pneumonia.
Bronchoalveolar lavage (BAL), as a standard diagnostic procedure for evaluating the in ammatory and immune processes of the lung infectious diseases, reveals the process of lung diseases and e cacy of treatment after lower respiratory tract infections (Smith 2015 ; Costable and Guzman 2001).
Macrophages and neutrophils, as dominant immune responders in the defense system of ruminants, especially cattle exert protective effects against various infections. Following the experimental pneumonia, neutrophil levels increased, as determined by BALF assessment. While after vitamin D 3 injection, the number of macrophages increased and that of neutrophils decreased in comparison to the control group.
The correlations between reduced pulmonary in ammation as determined by BALF cell counts and increased expression of Defensin and CD4 genes subsequent to vitamin D 3 intake were proved in this study.
Considering the leading roles of Cathalicidin and Defensin in pulmonary disease and the correlation between their expression and vitamin D 3 level, it could be suggested that vitamin D 3 de ciency may exacerbates lung problems.
In other words, increased vitamin D 3 level improves the expression of AMPs, especially Defensin, as well as improving lung function during pneumonia.
Consequently, it can be assumed that during the corona virus outbreak, vitamin D 3 intake may exert and positive effects on immune system through the expression of AMPs and regulation of cellular functionalities in the lung; thereby, improving the lung function and reducing the pulmonary impairment.
In both animal models and human cell lines, vitamin D 3 affected lung immunity and revealed protective effects on experimental interstitial pneumonia in this study. Numerous in vitro studies have indicated the considerable contribution of vitamin D 3 to local "respiratory homeostasis" one or the other by stimulating the expression of During the pneumonia outbreak, it seems de ciency of this vitamin can reduced the expression of AMPs, and subsequently lead to the increment in the rate of lung dysfunction. Therefore, su cient levels of this vitamin can remarkably reduce the severity of pulmonary symptoms caused by various bacteria.

Conclusion
Important functions of vitamin D 3 have been understood in calcium metabolism and phosphate homeostasis, activation of VDR pathways, and production of antimicrobial peptides (AMPs) for supporting the immune system. According to the results of this study, the immunomodulatory effects of vitamin D 3 and accordingly the signi cant improvement was observed in the clinical symptoms and increment of expression of AMPs especially lung AMPs in the in vitro and in vivo situations were proved. Therefore, given the major role of vitamin D 3 in improving the function of AMPs as well as in ammatory and pro-in ammatory cytokines, it is recommended that this vitamin should be used along with common antibiotic treatments to improve the healing process and decrease the frequency and severity of respiratory diseases.