On the Action of Cyclosporine A, Rapamycin and Tacrolimus on M. avium Including Subspecies paratuberculosis

Background Mycobacterium avium subspecies paratuberculosis (MAP) may be zoonotic. Recently the “immuno-modulators” methotrexate, azathioprine and 6-MP and the “anti-inflammatory” 5-ASA have been shown to inhibit MAP growth in vitro. We concluded that their most plausible mechanism of action is as antiMAP antibiotics. The “immunosuppressants” Cyclosporine A, Rapamycin and Tacrolimus (FK 506) treat a variety of “autoimmune” and “inflammatory” diseases. Rapamycin and Tacrolimus are macrolides. We hypothesized that their mode of action may simply be to inhibit MAP growth. Methodology The effect on radiometric MAP 14CO2 growth kinetics of Cyclosporine A, Rapamycin and Tacrolimus on MAP cultured from humans (Dominic & UCF 4) or ruminants (ATCC 19698 & 303) and M. avium subspecies avium (ATCC 25291 & 101) are presented as “percent decrease in cumulative GI” (%-ΔcGI.) Principal Findings The positive control clofazimine has 99%-ΔcGI at 0.5 µg/ml (Dominic). Phthalimide, a negative control has no dose dependent inhibition on any strain. Against MAP there is dose dependent inhibition by the immunosuppressants. Cyclosporine has 97%-ΔcGI by 32 µg/ml (Dominic), Rapamycin has 74%-ΔcGI by 64 µg/ml (UCF 4) and Tacrolimus 43%-ΔcGI by 64 µg/ml (UCF 4) Conclusions We show heretofore-undescribed inhibition of MAP growth in vitro by “immunosuppressants;” the cyclic undecapeptide Cyclosporine A, and the macrolides Rapamycin and Tacrolimus. These data are compatible with our thesis that, unknowingly, the medical profession has been treating MAP infections since 1942 when 5-ASA and subsequently azathioprine, 6-MP and methotrexate were introduced in the therapy of some “autoimmune” and “inflammatory” diseases.


Methods
This study was approved by the Research & Development Committee at the VAMC Bronx NY (0720-06-038) and was conducted under the Institutional Radioactive Materials Permit (#31-00636-07).

Bacterial Culture
In this study we studied six strains of mycobacteria, four of which were MAP. Two MAP strains had been isolated from humans with Crohn's disease. Dominic (ATCC 43545, originally isolated by R. Chiodini from the intestine of a patient with Crohn's disease [48]) and UCF 4 (gift of Saleh Naser UCF Orlando FL., originally cultured from the blood of a patient with Crohn's disease.) [32] The other two MAP strains were from ruminants with Johne's disease ATCC 19698 (ATCC Rockville MD) and 303 (gift of Michael Collins Madison WI.) The M. avium subspecies avium strains (hereinafter called M. avium) were ATCC 25291 (veterinary source) and M. avium 101. [49] Because it renders clinically resistant strains of MAP inappropriately susceptible to antimicrobials in cell culture, [50] we did not use the detergent Tween 80 (recommended to prevent mycobacterial clumping) in culture. Prior to inoculation, cultures were processed as described. [34,37,51] In this study, for experimental comparability we used chemicals that could be solubilized with DMSO (Sigma St Louis MO.) The positive control antibiotic was clofazimine (an antibiotic used to treat leprosy [52] and now in clinical trials against Crohn's disease [39,53].) The two negative controls are the gluterimide antibiotics, cycloheximide and phthalimide.
The tested agents Cyclosporine A, Rapamycin and Tacrolimus (Sigma & LC Labs. Woburn MA) were solubilized in 100% DMSO. Aliquots were prediluted, stored at 280uC in 50% DMSO (Sigma) & 50% water, thawed, used once and discarded. Volumes of DMSO were adjusted so that final concentration in every Bactec vial used was always 3.2% DMSO. Agents were tested in serial dilutions from a minimum of 0.5 mg/ml to a maximum of 64 mg/ml (see individual Figures & Tables). Inhibition of mycobacterial growth is expressed as % -DcGI, and enhancement as % +DcGI compared to 3.2% DMSO controls. [37] Data are presented in two ways: For individual mycobacterial strains as graphs (MAP in Figures 1& 2, and M. avium in Figure 3.) For individual chemical agents data are presented in tabular form. The positive experimental control is clofazimine ( Table 1.) The ''negative'' controls are cycloheximide (Table 2) and phthalimide (Table 3.) Data for the ''immunosuppressives'' are Cyclosporine A (Table 4), Rapamycin (Table 5) and Tacrolimus (Table 6.) In Table 7 we present the ''High'' trough doses of the three immunosuppressives that are used to treat organ transplant rejection in eukaryotes. These are compared with the ''Low'' dose that are used to treat ''inflammatory'' diseases and that we posit are actually treating a prokaryote (specifically we suggest a MAP) infection.

Results
The most potent positive control is clofazimine, 97% 2DcGI at 0.5 (Dominic; Figure 1 & Table 1.) The negative controls chemical agents are the gluterimide antibiotics cycloheximide and phthalimide. Cycloheximide has no dose dependent inhibition on any MAP strain (Figures 1 & 2 & Table 2.) Cycloheximide has dose dependent inhibition on M. avium ATCC 25291, (57% 2DcGI at 64 mg/ml) but no effect on M. avium 101 (Figure 3 & Table 2.) Phthalimide, has no dose dependent effect on any strain tested (Figures 1-3 and Table 3.) The three ''Immunosuppressants'' tested were Cyclosporine A, Rapamycin and Tacrolimus. There are differing amounts of inhibition depending on the agent and strain.
The control mycobacterial strains are M. avium subspecies avium ATCC 25291 and 101. Of the three ''Immunosuppressants,'' Cyclosporine A has dose dependent inhibition on M. avium subspecies avium 101 (95% 2DcGI at 64 mg/ml) (Figure 3 and Table 4.) There is no inhibition with Rapamycin or Tacrolimus on the control M. avium 25291 (Figure 3 and Table 5 & 6.) Against MAP, Cyclosporine A is the most effective of the three ''immunosuppressants'' studied. On MAP isolated from humans, (Dominic and UCF 4), Cyclosporine has 97% 2DcGI at 32 mg/ml against Dominic ( Figure 1) and 99% 2DcGI at 64 mg/ml on Dominic and UCF 4 ( Figure 2 & Table 4.) On MAP isolated from ruminants, Cyclosporine A has slightly less dose dependent   Table 5). Rapamycin is less effective against MAP isolated from ruminants and has no effect on M. avium ATCC 25291 (Figure 3 & Table 5.)           Tacrolimus has the least inhibition of the three ''immunosuppressants'' studied. Against MAP, Tacrolimus is most inhibitory against UCF 4 (43% 2DcGI at 64 mg/ml) and ATCC 19698: 26% 2DcGI at 64 mg/ml) (Figures 1 & 2 and Table 6.) Paradoxically, Tacrolimus exhibits the most inhibition on M. avium 101 of all six strains studied, yet actually enhances growth on M. avium ATCC 25291. (Figure 3 and Table 6.)

Discussion
Rapamycin was initially evaluated as an anti-fungal agent. [54] To our knowledge however, this is the first time that antiMAP activity has been demonstrated for the ''immunosuppressant'' agents Cyclosporine, Rapamycin and Tacrolimus. These observations are therefore compatible with our thesis that MAP may be responsible for multiple ''autoimmune'' and ''inflammatory'' diseases, and that the action of these three ''immunosuppressant'' agents may simply be to inhibit MAP growth.
We have observed that methotrexate and 6-MP are used in ''high'' doses to treat human malignancies and at ''low'' doses in ''autoimmune'' and ''inflammatory'' conditions. [34] Similarly, there are ''high'' and ''low'' doses of the three ''immunosuppressants'' we now study (See Table 7.) The ''high'' doses are used to prevent or treat transplanted organ rejection. The ''low'' doses are used to treat ''autoimmune'' and ''inflammatory'' diseases. These data are compatible with our hypothesis that Cyclosporine, a cyclic undecapeptide, as well as Rapamycin and Tacrolimus, from the macrolide family of antibiotics, may have ''low'' dose prokaryotic antibiotic action in addition to ''high'' dose eukaryotic immunosuppressant activity.
Our observations are subtle and the negative controls are critical. For those not conversant with quantifying mycobacterial growth and determining the inhibitory effect of various agents, it must be emphasized that these data were obtained using the exquisitely sensitive radiometric 14 C Bactec systemH. Just as with 5-ASA [37,38], these effects may not be detectable using the more convenient, fluorescent based MIGT systemH.
The chronic use of antibiotics, even for complex mycobacterial diseases, is not advocated. With leprosy the WHO recommends that MDT be limited to #2 years [52] and for tuberculosis #18 months and preferably six months. [55] The ''immunosuppressant, '' ''antiinflammatory'' and ''immunomodulatory'' agents that we show are antiMAP antibiotics have been administered indefinitely.
In the event that MAP is accepted as being zoonotic, there will need to be a reevaluation of how best to manage MAP infections in humans. There will be multiple factors that will then need to be taken into consideration. These include the fact that successfully treated leprosy and tuberculosis infections do not lead to mycobacterial eradication. Often the bacteria merely enter into a quiescent or ''latent'' phase and clinical symptoms progress [56] despite apparently ''adequate'' therapy. It will also be necessary to prevent reinfection, by removing MAP from the water supply [27], and food chain. [28] Genetic defects [57][58][59] that predispose to MAP infections will need to be identified, as affected individuals may need life long antiMAP therapy. Optimal MAP antibiotic combinations will need to be established. Designing clinical trial that consider the recently described antiMAP activity of ''antiinflammatories'', ''immunomodulators'' and ''immunosuppressants'' will need to be performed. Finally, the role of MAP pre and post exposure vaccination will need to be addressed. [60][61][62]