Gut microbiota and graft-versus-host disease in hematopoietic stem cell transplant patients.

INTRODUCTION

Allogeneic hematopoietic stem cell transplantation (HSCT) is a highly effective immunotherapeutic modality that is widely acknowledged for its efficacy in treating a range of both malignant and non-malignant hematological conditions ( 1 ). Despite recent advances as a treatment option, patients are severely immunocompromised after HSCT. Meanwhile, These patients remain vulnerable to viral infections as well as graft-versus-host disease (GvHD), which constitutes a significant factor contributing to mortality ( 2 ).

GvHD is an important and common complication of allogeneic HSCT and can cause complications in 30–70% of allogeneic HSCT recipients and 15% post-transplant mortality ( 3 , 4 ). GvHD can be classified into two disease groups: acute (symptoms appearing before 100 days after allo-HSCT) or chronic (symptoms appearing > 100 days after allo-HSCT) ( 5 ). GvHD affects multiple organs in the body, particularly the gastrointestinal tract, liver, skin, eyes, and lungs. This condition is characterized by the damage to host tissues and organs, primarily mediated by immune cells, especially donor T cells. These T cells recognize and respond to major and minor histocompatibility alloantigens presented by antigen-presenting cells ( 3 , 6 ). Research involving both animal models and humans has demonstrated a significant correlation between gut microbiota and the risk of GvHD; however, this association remains ambiguous ( 3 ). Modifying gut microbiota offers a promising strategy for preventing or treating this common condition.

The gut microbiota comprises various microorganisms that inhabit the gastrointestinal (GI) tract, and this microbial community in an adult human consists of 10 13 to 10 14 CFU/ml microorganisms ( 7 , 8 ). The human intestinal microbiota is composed of a wide variety of microorganisms, including bacteria, archaea, fungi, protozoa, and viruses, with bacteria representing the predominant group among these organisms ( 9 ). In general, four phyla of bacteria including Firmicutes and Bacteroidetes (approximately 90) and Proteobacteria and Actinomycetes (with lower abundance) dominate the human gut microbiota. Recent studies indicate a correlation between specific arrangements of microbial communities and various health outcomes, including mortality rates, the recurrence of diseases, susceptibility to infections, and the incidence of GVHD ( 10 , 11 ). On the other hand, the microbiota may directly influence the risk of infection and shape or disrupt the microbial communities in the patients.

The release of inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6), during the conditioning regimen plays a critical role in T cell activation and substantially contributes to the development of graft-versus-host disease (GvHD) following transplantation ( 12 ). This study aims to investigate the microbiota in HSCT patients before and after transplantation and its association with individuals experiencing GvHD.

MATERIALS AND METHODS

Patients and specimens. This research was conducted from December 2022 to December 2023. 15 stool samples of HSCT patients were collected from the Bone Marrow Transplant Center of Imam Reza Hospital, Kermanshah, Iran. The specimens were appropriately labeled and preserved at −70°C until the process of DNA extraction. Also, blood samples were taken from patients one week after transplantation and their serum was stored at −70°C. Stool samples were collected from these patients on three occasions (before transplantation, one week, and one month after transplantation). The reason for choosing one week and one month of sampling after transplantation was the initiation of the inflammatory phase, which typically occurs between three to seven days post-allo-HSCT transplantation, as well as the subsequent emergence of GvHD complications ( 13 ). The age range of patients with HSCT was between 21 to 65 years.

The inclusion and exclusion criteria of this study are allo-HSCT and auto-HSCT patients, respectively, because GvHD occurred only in patients who underwent allo-HSCT.

DNA extraction. DNA was extracted from stool samples using the FavorGen extraction kit (Pintung, Taiwan) according to the manufacturer’s instructions and stored at −20°C. The evaluation of DNA quality was performed using an ultraviolet spectrophotometer (Nanodrop Technologies, Inc., Wilmington, DE, USA), measuring at wavelengths of 260 and 280 nanometers.

Real-time PCR. The Light Cycler system (Roche) was utilized to perform the real-time PCR assay. Amplification reactions were performed in a volume of 20 μl containing 5 μl of DNA (20 ng/µl) template, 0.5 μL each for forward and reverse primers (10 pmol/µl), 10 μl of SYBR Green PCR Master Mix (Ampliqon UK) and 4 μl of sterile double distilled water. The primer sequences in this study were developed using Oligo 7 software, ( 14 ) (Molecular Biology Insights, Inc., Cascade, CO, USA) and the OligoAnalyzer online server https://www.idtdna.com/pages/tools/oligoanalyzer ). These primers were subsequently synthesized by Metabion in Germany. Furthermore, the alignment of the primers was conducted through multiple sequence alignment (MSA) methods using Clustal Omega software ( https://www.ebi.ac.uk/Tools/msa/clustalo/ ). The sequences of the designed primers are detailed in Table 1 . The real-time PCR temperature program is outlined in Table 2 . The real-time PCR assay was performed in duplicate to ensure accuracy.

Enzyme-linked immunosorbent assay (ELISA). Enzyme-linked immunosorbent assay (ELISA) is widely a significant method for the serodiagnosis of infectious diseases ( 15 ). To measure Interleukin 6 (IL-6) of patients after transplantation, their serum samples, which were centrifuged after 10 minutes at 4°C and 3000 r/min, were used to perform the ELISA test. In this study, a kit (Zellbio, Germany) was used to detect IL-6 in serum samples. Briefly, combinations (40 μl samples + 10 μl anti-IL-6) and 50 μl standard with 50 μl streptavidin-HRP in 96-well flat plates, were incubated at 37°C for 60 minutes. Then the plate was washed five times with 300 μl of diluted wash buffer. 100 μl of chromogen solution was added and incubated at room temperature for 10 to 20 minutes. Subsequently, 50 μl of stop solution was added and its OD was measured at wavelength 450 nm.

Statistical analysis. The data were analyzed by IBM SPSS statistics® Version 25 (IBM Corp, Armonk, NY, USA). Friedman tests were used to estimate the significant difference between group comparisons. P value ≤ 0.05 was considered statistically significant in comparative data.

RESULTS

From a total of 15 (8 male and 7 female) HSCT patients, 9 and 6 allo-HSCT and Auto-HSCT patients were included in our study, respectively. Among allo-HSCT patients, 4 patients were full-matched and 5 patients were Haplo-HSCT. On the other hand, out of a total of 15 patients, 40%, 13.33%, and 46.67% had multiple myeloma, aplastic anemia and acute myeloid leukemia (AML), respectively. GvHD occurred only in patients who underwent allo-HSCT. In other words, out of 9 allo-HSCT patients, 4 (44.44%) patients became GvHD.

Evaluation of ELISA test. The purpose of the ELISA test was to determine whether interleukin-6 could be useful as a prognostic factor for GvHD. The results of this study showed that all our samples were reported within the normal range (30–960 pg/ml) and no significant increase in interleukin-6 levels was observed in GvHD in HSCT patients.

Prevalence of bacteria in allo-HSCT patients. The alterations in microbiota diversity were investigated in both the GvHD and non-GvHD groups ( Tables 3 and 4 ; Figs. 1 and 2 ). In the evaluation of the genus Bacteroides in the GvHD group, the patients experienced a reduction in the bacterial count between the before the transplant and the intervals of one week and one month following the transplant. (P-value≤0.05). On the other hand, in the non-GvHD group, no significant difference was observed before and after transplantation. In Bifidobacterium in the GvHD group, the number of bacteria decreased one week and one month after transplantation compared to patients before transplantation (P-value=0.013). In the non-GvHD group, no significant difference was observed before and after transplantation.

Our research indicated a significant difference in the quantity of Clostridium bacteria in the GvHD group between before transplantation and one month after transplantation (P-value=≤0.05) and the number of bacteria decreased in this group. Also, in the group without GvHD, there was a decrease in the number of bacteria between one week and one month after transplantation, and there was a statistically significant difference (P-value≤0.05).

While no notable correlation was observed regarding Enterobacterials in the GvHD group before and after transplantation, the group without GvHD exhibited a significant increase in bacterial levels one-month post-transplantation when compared to one-week post-transplantation (P-value=0.018).

Actinobacteria increased in the group without GvHD between one month and one week after transplantation, but a significant difference (P-value=0.022) was observed in the group with GvHD.

Fusobacterium was significantly different in both GvHD and non-GvHD groups between one week and one month after transplantation. In addition, Blautia in the group without GvHD, there was no significant difference in Blautia levels observed between the pre-transplant and post-transplant stages, but in the GvHD group, it increased between before and one week after transplantation (P-value=0.034).

According to ( Tables 3 and 4 ), there was a significant difference in the levels of Lactobacillus and Firmicutes in the group that did not experience GvHD, with a marked increase in bacterial counts observed one-month post-transplantation compared to one week after the procedure. (Lactobacillus; P-value=0.004 and Firmicutes ; P-value=0.007). In GvHD patients, these bacteria decreased between before and one week after transplantation ( Lactobacillus ; P-value=0.034 and Firmicutes ; P-value=0.022).

DISCUSSION

Studies investigating the microbiota composition in patients before and after allo-HSCT have shown a decrease in bacterial diversity following treatment. This reduction in diversity was especially marked in patients with intestinal GvHD ( 10 ). Our findings from HSCT patients revealed significant alterations in the intestinal bacterial ecosystem. Compared with the pre-transplant microbiota, the gut microbiota following HSCT exhibited a reduction in bacterial diversity which is associated with an increased risk of infection, GvHD, and mortality following allo-HSCT. In the aGvHD group, a decrease in the number of Bacteroides and Bifidobacterium bacteria was evident compared to the group without aGvHD. A decrease in the number of Clostridium and Firmicutes bacteria was also reported in aGvHD group. Another notable result was the genus Blautia , which was present and reduced in the aGvHD group, but no significant difference in the number of bacteria was observed in the uncomplicated group. In addition, the number of Actinobacteria, Lactobacillus and Fusobacterium increased in the aGvHD group.

Consistent with our study, Doki et al analyzed stool samples from approximately two weeks before the conditioning phase in recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Their results indicated that patients with aGvHD exhibited a markedly increased prevalence of Firmicutes while demonstrating a reduced occurrence of Bacteroides in comparison to patients without aGvHD ( 16 ). In another study, Li et al. analyzed the stool samples of 11 patients to study the changes in intestinal microbiota following allo-HSCT. The results indicated that the diversity of intestinal microbiota in patients with aGvHD was significantly reduced compared to those without aGvHD, both before and after to the transplantation ( 17 ). Similar to the mentioned study, we also saw a decrease in the number and diversity of intestinal microbiota using specific primers. In another study, Kouidhi et al investigated changes in gut microbiota composition in stool samples using Next-generation sequencing (NGS). Their results were associated with the abundance of Actinobacteria, Firmicutes, and Bacteroides after allo-HSCT ( 18 ). However, we observed an increase in Actinobacteria and a decrease in Firmicutes , and Bacteroides between one week and one month after transplantation.

In addition, our results showed that a reduction in microbial diversity within the intestinal tract, specifically the genus Blautia following hematopoietic stem cell transplantation (HSCT), correlated with increased lethality from graft-versus-host disease (GvHD), which was similar to the study by Jenq et al. ( 19 ). Consistent with our results, other studies have indicated that the bacterial community experienced a reduction in Clostridia following allogeneic hematopoietic stem cell transplantation (allo-HSCT) ( 18 , 20 ). Studies have shown that a conditioning regimen including radioactive sources may result in dysbiosis and alterations in the microbiota ( 21 ).

Evidence indicates that both pro-inflammatory and anti-inflammatory cytokines play a significant role in influencing the risk of acute graft-versus-host disease (aGvHD) so IL6 can be associated with several inflammatory diseases ( 22 ). Interleukin-6 (IL-6) has the potential to provide benefits for individuals at increased risk of developing acute graft-versus-host disease (aGvHD) ( 23 ). In other words, this cytokine is crucial for implementing preventive strategies aimed at enhancing the quality of life during the initial phase following transplantation and the outcomes of allo-HSCT. It should be noted that the main limitation of this study was the absence of next-generation sequencing (NGS) equipment.

CONCLUSION

Our results demonstrated that the presence of a specific bacterial group within the gut microbiota of transplant patients is associated with an increased incidence of transplant-related diseases among individuals undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). The study investigated changes in the diversity of gut microbiota in both groups with GvHD and those without it. In the aGvHD group, a decrease in the number of Bacteroides , and Bifidobacterium bacteria was evident compared to the group without aGvHD. Also, a decrease in the number of Clostridium and Firmicutes bacteria was seen, which was not significant in the other group. Another significant result was the genus Blautia , which was present and reduced in the aGvHD group, but no significant difference in bacterial counts was observed in another group. In addition, an increase in Actinobacteria and Fusobacterium genera was observed in the aGvHD group. It is important to highlight that a major limitation of this research was the absence of next-generation sequencing (NGS) technology.

ACKNOWLEDGEMENTS

This research was conducted in accordance with the Code of Ethics Committee (Reference ID: IR. KUMS. MED. REC.1401.159) established by Kermanshah University of Medical Sciences.

REFERENCES

1. Gupta V, Braun TM, Chowdhury M, Tewari M, Choi SW. A systematic review of machine learning techniques in hematopoietic stem cell transplantation (HSCT). Sensors (Basel) 2020; 20: 6100.

2. Qian C, Wang Y, Reppel L, D'aveni M, Campidelli A, Decot V, et al. Viral-specific T-cell transfer from HSCT donor for the treatment of viral infections or diseases after HSCT. Bone Marrow Transplant 2018; 53: 114–122.

3. Fredricks DN. The gut microbiota and graft-versus-host disease. J Clin Invest 2019; 129: 1808–1817.

4. Ramachandran V, Kolli SS, Strowd LC. Review of graft-versus-host disease. Dermatol Clin 2019; 37: 569–582.

5. Malard F, Holler E, Sandmaier BM, Huang H, Mohty M. Acute graft-versus-host disease. Nat Rev Dis Primers 2023; 9: 27.

6. Ferrara JL, Levine JE, Reddy P, Holler E. Graft-versus-host disease. Lancet 2009; 373: 1550–1561.

7. Turnbaugh PJ, Ley RE, Hamady M, Fraser-Liggett CM, Knight R, Gordon JI. The human microbiome project. Nature 2007; 449: 804–810.

8. Sędzikowska A, Szablewski L. Human gut microbiota in health and selected cancers. Int J Mol Sci 2021; 22: 13440.

9. Andermann TM, Peled JU, Ho C, Reddy P, Riches M, Storb R, et al. The microbiome and hematopoietic cell transplantation: past, present, and future. Biol Blood Marrow Transplant 2018; 24: 1322–1340.

10. Holler E, Butzhammer P, Schmid K, Hundsrucker C, Koestler J, Peter K, et al. Metagenomic analysis of the stool microbiome in patients receiving allogeneic stem cell transplantation: loss of diversity is associated with use of systemic antibiotics and more pronounced in gastrointestinal graft-versus-host disease. Biol Blood Marrow Transplant 2014; 20: 640–645.

11. Peled JU, Devlin SM, Staffas A, Lumish M, Khanin R, Littmann ER, et al. Intestinal microbiota and relapse after hematopoietic-cell transplantation. J Clin Oncol 2017; 35: 1650–1659.

12. Ferrara JL. Pathogenesis of acute graft-versus-host disease: cytokines and cellular effectors. J Hematother Stem Cell Res 2000; 9: 299–306.

13. Zeiser R, Blazar BR. Pathophysiology of chronic graft-versus-host disease and therapeutic targets. N Engl J Med 2017; 377: 2565–2579.

14. Rychlik W. OLIGO 7 primer analysis software. Methods Mol Biol 2007; 402: 35–60.

15. Zhou J, Chen J, Peng Y, Xie Y, Xiao Y. A promising tool in serological diagnosis: current research progress of antigenic epitopes in infectious diseases. Pathogens 2022; 11: 1095.

16. Doki N, Suyama M, Sasajima S, Ota J, Igarashi A, Mimura I, et al. Clinical impact of pre-transplant gut microbial diversity on outcomes of allogeneic hematopoietic stem cell transplantation. Ann Hematol 2017; 96: 1517–1523.

17. Li DS, Wu YR, Du WH, Zhu YL, Zhang WJ, Fu Y, et al. The composition of the intestinal microbiota after allogeneic haematopoietic stem cell transplantation and its association with graft versus host disease as assessed by 16Sribosomal ribonucleic acid. J Physiol Pharmacol 2023; 74: 10.26402/jpp.2023.1.10.

18. Kouidhi S, Souai N, Zidi O, Mosbah A, Lakhal A, Ben Othmane T, et al. High throughput analysis reveals changes in gut microbiota and specific fecal metabolomic signature in hematopoietic stem cell transplant patients. Microorganisms 2021; 9: 1845.

19. Jenq RR, Taur Y, Devlin SM, Ponce DM, Goldberg JD, Ahr KF, et al. Intestinal Blautia is associated with reduced death from graft-versus-host disease. Biol Blood Marrow Transplant 2015; 21: 1373–1383.

20. Vital M, Howe AC, Tiedje JM. Revealing the bacterial butyrate synthesis pathways by analyzing (meta) genomic data. mBio 2014; 5(2): e00889.

21. Metafuni E, Di Marino L, Giammarco S, Bellesi S, Limongiello MA, Sorà F, et al. The role of fecal microbiota transplantation in the allogeneic stem cell transplant setting. Microorganisms 2023; 11: 2182.

22. Narazaki M, Kishimoto T. The two-faced cytokine IL-6 in host defense and diseases. Int J Mol Sci 2018; 19: 3528.

23. Greco R, Lorentino F, Nitti R, Lupo Stanghellini MT, Giglio F, Clerici D, et al. Interleukin-6 as biomarker for acute GvHD and survival after allogeneic transplant with post-transplant cyclophosphamide. Front Immunol 2019; 10: 2319.