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Review: Therapeutic Effect of L. helveticus (SBT2171) on Collagen-Induced Arthritis

There is an oft quoted statistic that the number of bacteria living in and on an individual outnumbers their human cells (37.2 trillion cells) by a ratio of 10:1 (1). A recent report shows that while bacteria do outnumber human cells, the ratio is closer to 1:1 rather than 10:1 (2). Regardless of what the actual ratio may be, there are two things that are certain:

1) The human body harbors a staggering number and a diverse array of microbes.
2) These microbes and the proteins they produce are potent stimulators of the immune system.

While the first claim is nothing surprising, the implications of the second claim are only beginning to be understood. Immunological methods, including germ-free and specific pathogen-free disease models, have enhanced researchers ability to isolate specific bacteria to assess their roles in disease. By combining the insights provided from these models with advances in gene sequencing, researchers can sequence patients’ microbiomes and tease out the relationships between specific bacterial populations and disease states. For instance, segmented filamentous bacteria has been implicated in the K/BxN model of autoimmune arthritis (3) and the taxa Akkermansia has been linked with multiple sclerosis (4,5).

This emerging field of research has also allowed for the identification of disease attenuating bacterial species. One such species, Lactobacillus helveticus, has been previously shown by Hosoya et al. (2014) to attenuate Collagen-Induced Arthritis (CIA) in DBA/1 mice (6). This study, using Chondrex, Inc. manufactured Bovine Type II Collagen and Complete Freund’s Adjuvant, showed that intraperitoneal (i.p.) injection of heat killied L. helveticus significantly decreased arthritis incidence, arthritis severity, paw thickness, and body weight loss when compared to control mice.

You may be asking yourself why, out of the innumerable species of bacteria, the researchers chose L. helveticus (SBT2171)? L. helveticus, and other lactic acid producing bacteria, are used for fermentation of foods, such as in cheese making (including cheese produced by the company funding the study). Fermentation has been used by humans for centuries as a way to store food for long periods of time, before the invention of refrigerators and freezers. Even today fermented foods, such as kimchi and sauerkraut, remain staples of diets all over the world. Researchers are finding that these fermented foods, and the bacteria responsible for their fermentation, have positive effects on gastrointestinal health (7). 

In a follow up study to their 2014 study, Yamashita et al. (2017) sought to shed light on the mechanisms underlying the therapeutic effects of L helveticus. Again, they used the Collagen-Induced Arthritis model in DBA/1 mice to study the effects of oral feeding of live L. helveticus as compared to i.p. injection of heat killed L. helveticus. Briefly, CIA was induced in DBA/1 mice via injection of an emulsion consisting of Chondrex, Inc. manufactured Bovine Type II Collagen and Complete Freund’s Adjuvant on day 0 and day 21. At the same time, the experimental groups were either fed freeze dried L. helveticus or received an i.p. injection of heat killed L. helveticus. The control groups received feeding or i.p. injection of the vehicle only (NaHCO3/PBS and PBS, respectively), on the same schedule as the experimental groups. The full experimental protocol can be found in the original paper here.

After the second CIA immunization on day 21, Yamashita et al. began to record arthritis incidence and clinical arthritis scores, as well as hind paw thickening and body weight loss (both markers of arthritis progression) to see how L. helveticus affected arthritis onset. The oral feeding experimental group showed significant reduction in hind paw thickening and body weight loss when compared to controls. However, the overall incidence of arthritis and severity of arthritis was not affected by oral feeding of L. helveticus (the overall incidence was the same between the experimental group and control group by day 42). 

The i.p. injection experimental group also showed drastic reduction in hind paw thickening and body weight loss. In fact, this group actually gained weight, on average, over the course of the experiment. Furthermore, the i.p. injection of L. helveticus completely curtailed arthritis onset, with a 0% incidence and average clinical score of 0 for the mice in this group. Additionally, histological assessment of the joints in these mice on day 42 revealed that the cartilage damage seen in control groups was not found in the i.p. injection experimental group. 

To elucidate the immunological differences between the experimental groups, serum samples were collected from the CIA mice on day 42 and the anti-type II collagen total IgG, IgG1, and IgG2a titers were determined by ELISA. (They did use Chondrex manufactured ELISA Grade Collagen as the antigen for plate coating, but we also offer Mouse Anti-Type II Collagen Antibody Subtype Assay Kits!) Both experimental groups exhibited significant decreases in type II collagen specific IgG and IgG1 antibodies, but there was no significant difference in the levels of type II collagen specific IgG2a antibodies. Further analysis of CIA mice serum for IL-6 and TNFα levels by ELISA revealed that i.p. injection reduced IL-6 levels (but not TNFα levels), while oral feeding had no impact on these cytokine levels. 

Additionally, Yamashita et al. analyzed cells taken from the inguinal lymph nodes via flow cytometry in both the i.p. injection experimental and control groups. The number of B cells and CD4+ T cells were significantly lower in the experimental group than the control. More specifically, germinal center B cells were less abundant, while the plasma cell number was unaffected. Moreover, analysis of T helper cell types showed a reduction in the follicular T helper cell populations, but not regulatory T cells, Th1 or Th17 cells. 

So, what does this all really mean? Yamashita et al. offer a thorough literature review that outlines the importance of B cells, follicular T helper cells, and CD4+ T cells in the development of autoantibodies that are crucial for autoimmune arthritis. They argue that L. helveticus could reduce the proliferation of certain B cells and CD4+ T cells, therefore inhibiting autoantibody production at germinal centers. This low level of autoantibodies would be insufficient for complement fixation, resulting in low arthritis scores. Another possible explanation for the lack of autoantibody production is that i.p. injection of L. helveticus draws the attention of the inflammatory cells away the injected collagen, resulting in the attenuation of arthritis. This is similar to how CIA mice may have reduced arthritis severity if they are injured, experience high stress levels, or are housed in conventional conditions. 

While the i.p. injection results are consistent with previous work, the results of the oral feeding are not as expected. Previously, oral administration of Lactobacillus casei has been shown to exert suppressive effects on CIA in DBA/1 mice (8). However, in order for oral feeding to be effective, it is believed that there must be an accompanying change in the bacterial flora populations.  Therefore, further investigation into how Lactobacillus species can alter CIA progression is necessary and could lead the way towards effective treatment for autoimmune arthritis. 

This paper exemplifies the shift in autoimmune disease research towards identifying interactions between commensal bacteria, the host immune system, and disease states. Continued research in this field will lead the way to more effective treatments for complex autoimmune disorders. 

 

References

1) E. Bianconi, A. Piovesan, F. Facchin, A. Beraudi, R. Casadei, F. Frabetti, L. Vitale, M.C. Pelleri, S. Tassani, F. Piva, S. Perez-Amodio, P. Strippoli, S. Canaider. An estimation of the number of cells in the human body. Ann Hum Biol. 40(6):463-471,(2013). doi: 10.3109/03014460.2013.807878

2) R. Sender, S. Fuchs, R. Milo. Revised estimated for the number of human and bacteria cells in the body. PLoS Bio. 14(8):e1002533,(2016). doi: 10.1371/journal.pbio.1002533.

3) H.J. Wu, I.I. Ivanov, J. Darce, J. Hattori, T. Shima, Y. Umesaki, D.R. Littman, C. Benoist, D. Mathis. Gut-residing segmented filamentous bacteria drive autoimmune arthritis via T helper 17 cells. Immunity. 32(6):815-827,(2010). doi: 10.1016/j.immuni.2010.06.001.

4) E. Cekanaviciute, B.B. Yoo, T.F. Runia, J.W. Debelius, S. Singh, C.A. Nelson, R. Kanner, Y. Bencosme, Y.K. Lee, S.L. Hauser, E. Crabtree-Hartman, I.K. Sand, M. Garcias, Y. Zhu, P. Casaccia, B.A.C. Cree, R. Knight, S.K. Mazmanian, S.E Baranzini. Gut bacteria from multiple sclerosis patients modulate human T cells and exacerbate symptoms in mouse model. PNAS. 114(40):10713-17018,(2017).
doi: 10.1073/pnas.1711235114.

5) K. Berer, L.A. Gerdes, E. Cekanaviciute, X. Jia, L. Xiao, Z. Xia, C. Liu, L. Klotz, U. Stauffer, S.E. Baranzini, T. Kümpfel, R. Hohlfeld, G. Krishnamoorthy, H. Wekerle. Gut microbiota from multiple sclerosis patients enables spontaneous autoimmune encephalomyelitis in mice. PNAS. 114(40):10719-10724,(2017). doi: 10.1073/pnas.1711233114.

6) T. Hosoya, F. Sakai, M. Yamashita, T. Shiozaki, T. Endo, K. Ukibe, H. Uenishi, Y. Kadooka, T. Moriya, H. Nakagawa, Y. Nakayama, T. Miyazaki. Lactobacillus helveticus SBT2171 inhibits lymphocyte proliferation by regulation of the JNK signaling pathway. PLos One. 9(9):e108360,(2014). doi: 10.1371/journal.pone.0108360.

7) H. Jensen, S. Grimmer, K. Naterstad, L. Axelsson. In vitro testing of commercial and potential probiotic lactic acid bacteria. Int J Food Microbiol. 153(1-2):216-222,(2012). doi:10.1016/j.ijfoodmicro.2011.11.020.

8) I. Kato, K. Endo-Tanaka, T. Yokokura. Suppressive effects of the oral administration of Lactobacillus casei on type II collagen-induced arthritis in DBA/1 mice. Life Sci. 63(8):635-644,(1998).

 

Original Paper:
M. Yamashita, K. Matsumoto, T. Endo, K. Ukibe, T. Hosoya, Y. Matsubara, H. Nakagawa, F. Sakai, T, Miyazaki. Preventive effect of Lactobacillus helveticus SBT2171 on collagen-induced arthritis in mice. Front Microbiol. 8:1159,(2017). doi: 10.3389/fmicb.2017.01159