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Adv Biomed Res 2023,  12:183

Immuno-modulatory effects of inactivated Dietzia Maris on the selected aspects of cellular and humoral immune responses in mice

1 Department of Pathobiology, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran
2 Department of Basic Sciences, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran

Date of Submission18-Apr-2022
Date of Acceptance14-Feb-2023
Date of Web Publication20-Jul-2023

Correspondence Address:
Dr. Katayoon Nofouzi
Department of Pathobiology, Faculty of Veterinary Medicine, University of Tabriz, Tabriz - 51368
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/abr.abr_121_22

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Background: The current study is an attempt to register the alterations in the immunological and histological parameters in mice arising from the administration of Dietza maris (D. maris) in order to confirm its protective properties.
Materials and Methods: Mice underwent 7 days of treatment with three doses of D. maris. Then, animals were scrutinized in terms of body weight, relative weight of organs, delayed type of hypersensitivity (DTH) response, and hemagglutination titer (HT). The determination of villus height, villus width, crypt depth, villus/crypt ratio (V/C), Goblet cells, and intestinal epithelial lymphocyte (IEL) density in villi was carried out.
Results: A boosted DTH response was observed as a result of bacteria at medium dose. A variation was noted between the hemagglutinin titer of the control group and that of the high-dose group. Crypt depth, villus width, and villus height manifested alterations. High-dose-treated mice demonstrated proliferation of Goblet cells in the villi, whereas both in medium- and high-dose-treated mice, a distribution of IELs in the villus epithelium was noted. Overall, D. maris showed a stimulatory effect on immune functions in mice. Thus, thanks to improved cellular and humoral immunity and the increased quality of intestine function, we believe that D. maris promises novel therapeutic applications in the future.
Conclusion: The attained findings lend credence to immuno-stimulatory effects arising from the capacity of D. maris to function as immunological adjuvants and to enhance humoral and cellular immunity as well as the intestinal structure and function.

Keywords: Actinomycetales, Dietzia maris, immuno-modulation, intestines

How to cite this article:
Nofouzi K, Hamidian G. Immuno-modulatory effects of inactivated Dietzia Maris on the selected aspects of cellular and humoral immune responses in mice. Adv Biomed Res 2023;12:183

How to cite this URL:
Nofouzi K, Hamidian G. Immuno-modulatory effects of inactivated Dietzia Maris on the selected aspects of cellular and humoral immune responses in mice. Adv Biomed Res [serial online] 2023 [cited 2023 Sep 26];12:183. Available from:

  Introduction Top

On the basis of recent findings, the injection of a newly identified group of aerobic Actinomycetales species, such as Gordonia bronchialis (G. bronchialis), Rhodococcus coprophilus (R. coprophilus), and Tsukamurella inchonensis (T. inchonensis), which are quite comparable to mycobacteria, can bring somewhat various adjuvant or immuno-modulatory activities into play.[1] The application of such species has considerably amplified immune responses, yielded satisfactory intestinal function,[2] and diminished inflammatory responses in mice.[3]

In the beginning stages of life, human immunity is constantly regulated by myriad environmental agents which run the gamut from exposures to abiotic chemicals and nutritional conditions to biotic stressors originating from infectious diseases that are characterized by microbial or parasitic colonization.[4] However, scant evidence supports the fact that boosted immunity can reinforce resistance to infection in healthy individuals.

One of the most outstanding in vivo indications of cell-mediated immunity is delayed type hypersensitivity (DTH). The outbreak and progression of DTH triggered by immunization or an infection, which is induced by intra-cellular parasites and mediated by antigen-specific T cells, are well manifested by the synthesis of activating and chemotactic cytokines, a rise in vascular permeability, and deployment of antigen non-specific effector cells to the location where the reaction occurs.[5]

In order to retain epithelial integrity as well as keeping the organism unharmed by the pathogens rife in the environment, the intestinal barrier comes into play as a pivotal defense mechanism that encompasses the mucous layer, anti-microbial peptides, secretory IgA, and the epithelial junction adhesion complex. The active engagement of probiotic bacteria in fortifying the intestinal barrier has been extensively investigated; despite this, the mechanisms at work still remain obscure.[6] Any induction of damage instigated by various pathological agents, such as E. coli-induced mucosal disruption, prompts probiotics to set the repair of the barrier function into motion.[7] Probiotics are also capable of staving off any cytokine-induced epithelial damage, which is typical to inflammatory bowel disease.[8]

Intestinal morphology is a means to reveal any barrier function in the intestine. Intestinal function relies primarily on the heights and depths of the villi and crypt, respectively. Because the absorption of nutrients is contingent upon the size and surface area of the villi, larger villi ensure efficient uptake. On the other hand, any boost in digestion is determined by abundant crypt cells and deeper crypt and accordingly sufficient secretion of enzymes and overall efficient digestion and absorption.[9]

Dietzia maris (D. maris) is recognized as a Gram-positive, aerobic, mycolic acid-containing actinomycete, lacking aerial mycelium.[10] The former report divulged the satisfactory immuno-stimulatory function of T. inchonensis in addition to the structural modifications in the intestine mucosa of healthy suriyan mice induced by heat-killed T. inchonensis. A remarkable growth in the heights of villi and depths of crypts ensues the improvement of digestive efficiency and mucosal immunity.[2] Because our previous work mainly revolved around the inhibitory impacts of D. maris on nitric oxide production, the current work was thus devised to delve into other potentials of D. maris in reinforcing immune responses,[11] with a particular focus on the immunological and histological parameters in mice models affected by D. maris through assessing the interactions between laboratory parameters and various D. maris inoculum concentrations in mice. The findings contributed to gaining accurate insights into the relationships between the oral administration of D. maris and its clinical laboratory parameters from diverse angles.

  Materials and Methods Top

Strain preparation

We were gifted D. maris by BioEos Ltd., Kent, UK. Having grown in Sauton's medium, D. maris is then collected through centrifugation and finally washed in borate-buffered saline, pH 8.0. After confirming the standardized wet weight of the suspensions, borate-buffered saline was used for re-suspending them and eventually sterilization at 121°C was performed in an autoclave. Having undergone the heat treatment, major probiotic characteristics at the intestinal level remained intact in probiotic bacteria, with bacterial extracts and supernatants making up the majority and thus paving the way for developing preparations with minimum risk and more favorable pharmaceutical features (long shelf-lives, etc.).[8]


This study was performed on male albino laboratory mice (Mus musculus), with an age range spanning from 8 to 10 weeks and with a weight range of 20–22 g, which were provided by Pasteur Institute, Tehran, Iran. Laboratory conditions (temperature 25 ± 2°C; a photoperiod of 12 h light/12 h dark) were maintained at standard levels for all the animals. Commercial pellet diet and water were available ad libitum. The project complied with the ethical principles, the national norms, and the required standards for conducting Medical Research in Iran with approval ID: IR.TABRIZU.REC.1399.024.

Treatment protocol

As the initial stage of the in vivo study, animals were divided into four groups (I–IV), with each group encompassing minimum 15 animals. Group I (control) received normal saline; groups II, III, and IV received bacterial suspension containing 5 × 107, 1 × 108, and 2 × 108 cells of D. maris per mice, respectively.[11] The doses were administered by oral gavage on a daily basis for 7 successive days [Figure 1].
Figure 1: Schematic diagram showing the immunization steps of mice by Dietzia maris

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Body weight and organ weight

Right after 24 h following the last dose, the body weights of animals were measured and registered. An anesthetic overdose containing 40 mg/kg of ketamine 5% and 5 mg/kg of xylazine 2% was utilized to euthanize the mice, administered on an intra-peritoneal basis by expert personnel,[12] and then, the kidneys, testes, heart, liver, and spleen were dissected out and weighed. The relative weight of organs was calculated as the percentage ratio of body weight (organ weight (g) × 100/body weight (g)).

Assessment of humoral immune functions

On day 10 of the experiment, 0.2 mL of 10% chicken red blood cells (cRBCs)[11],[13] was utilized to challenge the animals within the experimental groups. The registered parameters of humoral immunity are listed below.

Hemagglutinin titer assay

The procedure suggested by Bin-Hafeez et al.[13] was followed for conducting the hemagglutinin titer assay. For serum preparation, blood was extracted from the hearts of five mice on the fifth day following the immunization with 0.2 ml of 10% CRBC. 50 μl of phosphate-buffered saline (pH 7.2) in 96-well microtiter plates was used for successive twofold dilutions of serum, and later, 50 μl of 1% CRBC suspension in phosphate-buffered saline was added. Subsequent to mixing, plates were stored at room temperature for 2 h. The highest serum dilution showing visible hemagglutination was set to be the indicator of the antibody titer.

Delayed type hypersensitivity response

The method of Sharififar et al.[14] delineated the DTH. The final day of the treatment with D. maris marked the subcutaneous immunization of five animals with 1 × 109 CRBC. Once again on the fifth day of immunization, all the animals were challenged via the injection of 1 × 108 cells in their left-hand footpad. Meanwhile, an equal volume of normal saline was injected to the right footpad to stave off trauma for non-specific swelling. The increase in footpad thickness was measured 24 h after the challenge using a digital caliper.

Histological and histo-morphometrical analysis

Subsequent to being challenged with D. maris on the 8th day, five animals from each group were euthanized by 40 mg/kg of ketamine 5% and 5 mg/kg of xylazine 2% via intra-peritoneal (ip) injection. After dissection, 5 mm of the terminal segment of the descending duodenum of each animal was fixed in 10% buffered neutral formaldehyde. Then, a standard embedding technique was utilized for tissue processing. Briefly, graded series of ethanol, xylene, and paraffin wax were deployed for dehydrating, clearing, and embedding of the tissue samples, respectively. In order for histological and histo-morphometrical analysis, tissue sections (5–7 μm) were stained with hematoxylin and eosin (H and E) and periodic acid Schiff (PAS) prior to the examination under a light microscope. Following the guidelines recommended by Oliveira-Sequeira et al.,[15] 10 crypt units of five sections per animal were probed for villus height and crypt depth as a requisite for histo-morphometrical study. Unlike the villus length, which was measured from the villus tip to the villus–crypt junction, the depth of the invagination between two villi was regarded as the crypt depth, which was carried out using a calibrated ocular micrometer. The villus/crypt (V/C) ratio was calculated by the villus height divided by crypt depth.

In order for evaluation of intra-epithelial lymphocyte (IEL) density in duodenum, the number of IEL/100 enterocytes in five well-oriented duodenal villi from each section was counted.[10] The lymphocytes above the basal membrane of enterocytes, considered as IELs, were scrutinized for their predominant distribution within the villous base, tip, and body as well as their location (supra-nuclear and sub-nuclear) within the villus epithelium. Acid Schiff positive cells in 100 enterocytes of each villi present in at least five non-adjacent sections were targeted for the quantification of Goblet cells in PAS staining slides.

Statistical analysis

The results are expressed as mean ± SE (standard error of mean), and one-way ANOVA followed by Tukey's post hoc was performed by version 22 of SPSS software, with the significant difference being set at P value <0.05.

  Results Top

Effects of bacteria on body weight and relative weight of organs

Body and relative weight of organs were not responsive to treatment with inactivated D. maris [Figure 2]. Mortality was not observed in any of the groups. Disparities in weight gain were not remarkable enough to be registered in various groups of animals. The treated animals demonstrated no noticeable recovery of spleen weight. The liver weight did not suffer a change as a result of D. maris application. Other major organs including kidney, testis, and heart of the animals belonging to the distinct groups did not suffer any marked drop in terms of relative organ weights.
Figure 2: Effect of heat-killed D. maris on body weight (g) and relative organ weight (%). (a) body weight (g); (b) spleen relative weight (%); (c) liver relative weight (%); (d) kidney relative weight (%); (e) testes relative weight (%); (f) heart relative weight (%). Each value is mean ± SEM of five individual mice in each group. High dose: Given 2 × 108 of bacteria Medium dose: Given 1 × 108 of bacteria; Low dose: Given with 5 × 107 bacteria

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Effect of bacteria on cell-mediated immunity parameters

A remarkably (P < 0.05) boosted DTH response [Figure 3] was the upshot of the administration of the inactivated bacteria at medium dose compared to the control animals, but there is no significant difference between low- and high-dose groups compared to the control group.
Figure 3: Effects of various doses of D. maris on the DTH response in mice, compared with the control group; High dose: Given 2 × 108 of bacteria; Medium dose: Given 1 × 108 of bacteria; Low dose: Given with 5 × 107 bacteria; A-bColumn with different letters was significantly different, when compared with the control animals. (P < 0.05)

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Effect of bacteria on humoral immunity parameters

In contrast to the hemagglutinin titer value of 20 manifested by mice in the high-dose group, the titer of the control group was 8.00 ± 0.00, a significant difference indeed [P < 0.05; [Figure 4]].
Figure 4: Effect of different doses of heat-killed Dietzia maris on HT titer using cRBC as an antigen in mice 10 days pre-treatment Values are presented as mean ± SE of five mice. a-bColumn with different letters was significantly different, when compared with the control animals. (P < 0.05)

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Histological analysis

[Figure 5] illustrates the morphological modifications of the duodenal tunica mucosa as a result of the oral administration of the inactivated D. maris. The administration of the inactivated D. maris gave rise to improved mucosal integrity and clarity as compared to the control group.
Figure 5: Photomicrographs of mice duodenums in different groups treated by heat-killed D. maris (Periodic acid Schiff staining, × 200); Control group administrated PBS and low-, medium-, and high-dose groups administered 5 × 107, 1 × 108, and 2 × 108 of bacteria, respectively

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The villus height, crypt depth, villus width, and V/C ratio in the upper segment of small intestine of control and inactivated D. maris-treated mice are shown in [Figure 3]. Although crypt depth, villus width, and villus height underwent substantial alterations upon the delivery of inactivated D. maris compared to the control group, V/C ratio between the low-dose and control groups did not reveal a notable difference [Figure 6]. The Goblet cell density rose as a consequence of the oral gavage delivery of inactivated D. maris, as revealed by histo-morhometrical analysis. In a similar manner, IELs proliferated in a dose-dependent mode. Although the villi experienced a marked rise in the numbers of Goblet cells (P < 0.05) in high-dose-treated mice [Figure 6], lamina epithelialis of the villus depicted a higher distribution of IELs (P < 0.05) in both medium- and high-dose-treated mice [Figure 6].
Figure 6: Effect of various doses of inactivated D. maris on villus height and width (a and b), crypt depth (c), villus to crypt ratio index (d), Goblet cells (e), and IEL density (f) in duodenum of mice; Data presented as mean ± SEM of five individual mice in each group. Control group administrated PBS and low-, medium-, and high-dose groups administered 5 × 107, 1 × 108, and 2 × 108 of bacteria, respectively. Columns with different letters were significantly different (P < 0.05)

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  Discussion Top

Changes in organ weights can be attributed to the toxicities induced by a test substrate. Unlike other studies aimed at assessing T. inchonensis and G. bronchialis in rainbow trout,[16],[17] where noteworthy contrasts were observed in relative weights of organs among the treated groups and between all the treated groups and the control, in the current study, organ (spleen, liver, kidney, testis, heart) and body mass did not alter, indicating that three doses of D. maris did not exert any detrimental effects on any of the organs, an observation which was further backed by histopathologic studies in these organs.

Because parameters of both humoral and cellular immunity evince stimulatory effects, the possession of such traits was proved by this study for D. maris in mice. As revealed by the hemagglutinin titer test, it was only at a high dose of 2 × 108 cells/mouse/day that the increased response elicited by bacteria was meaningful, despite the fact that all the experimental doses instigate this rise although not significantly. Thus, D. maris is believed to be potentially capable of influencing and modulating immune reactions to “self” antigens such as heat shock proteins as well as bolstering immunity against invasive bacterial challenges.[18] Hence, 2 × 108 cells/mouse/day is regarded as the finest immunity-stimulating dose in mice. Contingent on the nature of the antigen and its ability to trigger immune responses, the systemic antibody response can be reinforced dramatically by probiotic bacteria.[19] Being secreted by B cells, natural serum IgM is in charge of a broad array of functions; bacterial antigens such as lipopolysaccharides and capsular polysaccharides get trapped by such antibiotics with low affinity though [20]; moreover, natural IgM antibodies prompt the stimulation of IgG exclusive to bacterial and viral antigens.[21]

The medium dose of bacteria (1 × 108 cells/mouse/day) yielded the highest DTH response, which is normally regarded as the indicator of cell-mediated immunity. The mechanism underlying the surge in DTH during the cell-mediated immunity response can possibly spark off sensitized T-lymphocytes. Transformation to lymphoblasts and secretion of substances such as pro-inflammatory lymphokines ensue being challenged by antigen which culminates in the flocking of scavenger cells to the reaction spot.[14] The stimulatory function of D. maris to ignite the expression of the reaction is further proved by the soaring level of DTH response.[22] A boost in the immunity functions relies on Actinomycetes which effectively raise the quantity of cytokines,[23] with some of them exerting anti-inflammatory effects.[2],[11]

Both humoral and cellular arms of the immune system can be stimulated by means of D. maris. Owing to its function, LPS-induced production of pro-inflammatory cytokine of interleukin-6 (IL-6) can depict a descending trend in mice.[11] The immuno-stimulatory influences of two other Actinomycetes of G. bronchialis and T. inchonensis have been affirmed by a considerable body of evidence. The administration of T. inchonensis brought about a marked disparity in the measurement of IL-4 and INF-α in the serum of quails.[24] A fundamental soar in DTH response at a dose of 2 × 108 cells/mouse/day in mice can be triggered by this bacterium. The serum level of IL-6 and TNF-α exhibits noticeable variations in a dose-dependent manner as a consequence of the oral administration of Actinomycetales species in diabetic rats.[3]

Duodenum villus height and width showed a meaningful growth in height in all doses of inactivated D. maris-treated mice, euthanized 24 h after the final treatment; nevertheless, medium and high doses of bacteria gave rise to a fundamentally higher crypt depth and V/C ratio. Identical outcomes were attained by Nofouzi et al.[2] through the administration of inactivated T. inchonensis and also by Yang et al.[9] via the oral administration of live Bifidobacterium. The number of epithelial cells can determine the heights of the finger-like intestinal villi; in other words, taller villi represent masses of mature epithelial cells and enhanced intestinal absorption.[9] All groups manifested a remarkable increase in villus widths, with all these findings giving credence to the instrumental role of bacteria in expanding the villus surface and eventually refining nutrient absorption in the small intestine.

Crypt depth was immensely impacted by both medium and high doses of bacteria. When heightened proliferation of crypt cells teams up with diminished apoptosis, an elevated villus height appears. The growth status of the intestinal membrane is reflected in the innate capacity of the crypt cells for proliferation. Alterations in crypt depth can be manifested in the rate of crypt cell division and consequently the rate of digestion in the small intestine.[25]

The status of the small intestine is revealed by calculating the ratio of villus height to crypt depth (V/C); although feeding with D. maris elevated this ratio in mice compared to the control group, the enhancement was meager in low and medium groups, which reflects the positive change in crypt depth.

On the basis of our histo-morphometrical findings, a marked growth in the quantity of mucous Goblet cells and IELs in the lamina epithelialis of duodenal villi was yielded owing to the oral administration of inactivated D. maris. Mucous Goblet cells are ubiquitous along the entire length of the intestine, helping the formation of a mucous layer by secreting mucins. On one hand, thanks to this layer, the epithelium is safeguarded against potentially damaging antigens and molecules, and on the other hand, it functions as a lubricant for intestinal motility.[26] Infection breaks out upon the first encounter of intestinal bacteria with the mucous, followed by the penetrating activities of pathogens to access the epithelial cells. The degradation of mucous occurs via multiple strategies by microorganisms, including the reduction of mucin disulfide bonds (Helicobacter pylori), protease activity (Pseudomonas aeruginosa, Candida albicans, and Entamoeba histolytica), and glycosidase activity (mixed oral and intestinal microbial communities), for either invasive purposes or ingesting the mucous-derived nutrients.[27] The capacity of probiotic strains to amplify mucin secretion and to fortify the integrity of the mucosal barrier[28] can ultimately culminate in the enhancement of protective responses.[29] A marked hike in mucin secretion and accordingly boosted resistance of the intestinal mucosal membranes arose from the administration of inactivated D. maris at a dose of 2 × 108/mouse/day. Other studies regarded heightened mucous secretion as a highly likely mechanism deployed by probiotics to hone barrier function and deter pathogens.[26]

The fact that the oral administration of D. Maris also contributed to the soaring number of IELs in a dose-dependent manner begs the question whether the observed variations between D. maris and the control group concerning the distribution of IELs along the villi should be attributed to biological factors in the current study. A plausible deduction is that highly mature and developed enterocytes converge in the villus tip, possessing an optimal functional and morphological state, whereas the younger and underdeveloped enterocytes tend to accumulate in the villus base during a process of regenerative transformation, a process with speedier and more efficacious unfolding in the D. maris groups. There is only smattering and outdated information available concerning the normal range for IELs[30] that regulate intestinal epithelial surface integrity and homeostasis.[31] It is the presence of some antigens within the gut lumen rather than the antigens of the villous epithelial cells which spurs the IELs to grow in numbers;[30] thus, the reason behind the upsurge of IELs in our research can be assigned to the antigens of D. maris. In total conformity with our results, a large number of studies have disclosed a boom in lymphocyte populations, with IEL in particular in the wake of probiotic treatments.[3],[32],[33]

In essence, an intrinsic immunity pathway known as Toll like receptor-2 (TLR-2) enables probiotics to fine-tune the mucosal immune responses. This is the probable pathway D. maris makes use of as a Gram-positive bacteria.[34]

  Conclusions Top

Although digestion and absorption complications are typically attributed to defective gastro-intestinal tract, severe intestinal disorders could pose serious hazards to the intestinal protective and defense systems as well as bacterial/endotoxin translocation, all culminating in intestinal failure (IF). On the authority of our observations, intestinal tract function gets prone to the impacts instigated by D. maris via intestinal morphological alterations, which consequently ameliorates the efficiency and defensive function of the intestine. Although the exact modulatory mechanisms of Actinomycetes on intestinal function are yet to be known, there exist some speculations about the acceleration of crypt cell proliferation and growth and/or turnover of the intestinal epithelium. D. maris is assumed to exert its impact by making modifications to either deranged bowel flora or patients' tolerance to their own commensal flora and hence facilitating the pathogenesis of a host of GI disorders.

The findings of the current investigation need to be further complemented with more comprehensive studies focusing on the mechanisms of immuno-modulation of these bacteria and the prospective use in immuno-compromised individuals. Finally, we believe that D. maris promises novel therapeutic applications in the future, particularly as a cure for cancers.


The authors wish to express their gratitude to BioEos Ltd, Kent, UK, for providing the heat- killed D. maris.

Financial support and sponsorship

This study received financial support from the Research Affairs of the University of Tabriz.

Conflicts of interest

There are no conflicts of interest.

  References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]


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