1) The study examined the effects of infliximab treatment on Crohn's disease patients infected with Mycobacterium avium ssp. paratuberculosis (MAP), which has been associated with Crohn's disease. 2) Patients treated with infliximab showed significantly decreased levels of antibodies against two MAP proteins compared to untreated patients, suggesting infliximab decreases secretion of these proteins. 3) The study also found infliximab treatment was detrimental to the survival of MAP within infected macrophages, with significantly decreased survival of MAP in macrophages exposed to infliximab.

1. CROHNS-00471; No of Pages 2
Journal of Crohn's and Colitis (2012) xx, xxx–xxx
Available online at www.sciencedirect.com
LETTER TO THE EDITOR
Treatment of Crohn's disease patients with CD patients. A cohort of 20 CD patients treated with inflixi-
infliximab is detrimental for the survival of mab, 20 CD patients not treated with infliximab, and 20
Mycobacterium avium ssp. paratuberculosis within healthy controls were enrolled in this study. 43.3% of the
macrophages and shows a remarkable decrease in subjects were male with average ages for CD, infliximab,
and healthy control groups being 41 ± 14, 33 ± 12, and 46 ±
the immunogenicity of mycobacterial proteins
18 years, respectively. In the CD and infliximab groups, the
mean Harvey Bradshaw Index score was 5.2 ± 5.0 and 4.6 ±
4.5 and the mean time since diagnosis of CD was 10.4 ± 10.2
Dear Sir,
and 10.4 ± 8.2 years, respectively. The Research Ethics
Board of the University of British Columbia, Vancouver, Can-
ada, approved the protocol for this study. Protocols for
blood collection, serum processing, antigen production and
The association between Mycobacterium avium ssp. paratuber- ELISA were followed as published. 8
culosis (MAP) and Crohn's disease (CD) although debatable, is Patients treated with infliximab show a significant de-
supported by several studies 1 which have reported the detec- crease in the level of antibodies against both MAP proteins,
tion or isolation of MAP from human tissues 2 including serum, 3 and had levels similar to the negative control (Fig. 1), sug-
body fluids (breast milk), 4 and high levels of TNF-α was found gesting that inhibition of TNF-α has an effect in the secre-
secreted by the gut mucosa in MAP-associated CD patients. 5 tion of both mycobacterial proteins within CD patients.
Infliximab is a monoclonal antibody that specifically inhibits Next, we evaluated the survival of MAP in THP-1 cells trea-
TNF-α and is used as a current therapy for CD. ted with infliximab. Prior to the infection with MAP, 8 THP-
Recently, Nakase et al. 6 demonstrated that THP-1 cells 1 cells were differentiated with phorbol 12-myristate 13-
infected with MAP induced the production of a higher amount acetate and exposed to infliximab (5 μg/mL) for 4 and
of TNF-α when compared to macrophages infected with either
Mycobacterium avium or Mycobacterium smegmatis, suggest-
ing that MAP is directly involved in the upregulation of this
cytokine.
Previously, we have reported that MAP is able to infect, re-
side, and multiply intracellularly in human macrophages, 7,8
suggesting that the pathogen is able to subvert the host's im-
mune response to avoid its own demise even at the earliest
stage of the infection. Moreover, we have reported that CD pa-
tients, 8 but not healthy controls, have a significantly higher
level of antibodies against two MAP proteins, a Protein tyrosine
phosphatase (PtpA), and a Protein kinase (PknG). Both proteins
are part of the signal transduction system of the bacterium,
have been shown to be secreted within the host, and are essen-
tial for the intracellular survival and the establishment of a suc-
cessful infection of the MAP's close relative Mycobacterium
tuberculosis. 9,10 Therefore, to persist within the host, both
proteins have to be secreted in a regular manner by the patho-
gen, in order to manipulate the immunological response eli- Figure 1 Distribution of the antibody intensity readings. A box-
cited by macrophages. Recently, we have shown that CD plot analysis shows the distribution of the antibody intensity read-
patients possess a higher titer of antibodies against PtpA and ings obtained by ELISA in CD patients, CD patients under
PknG when compared to healthy controls, 8 and we suggested treatment with infliximab, and healthy controls (n = 20 individuals
that both proteins can be used to determine the status of in each category). *P-value obtained using Wilconox rank sum
MAP in CD patients. test. CD = Crohn's disease; PtpA = protein tyrosine phospha-
We report our analysis of the impact that infliximab upon tase; PknG = protein kinase G. Experiments were performed
the presence of antibodies against PtpA and PknG in sera of in triplicate.
1873-9946/$ - see front matter © 2012 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.crohns.2012.01.011
Please cite this article as: Bach H, et al, Treatment of Crohn's disease patients with infliximab is detrimental for the survival of Mycobacterium
avium ssp. paratuberculosis within macrophages..., Journal of Crohn's and Colitis (2012), doi:10.1016/j.crohns.2012.01.011

2. 2 Letter to the Editor
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Figure 2 Survival of MAP in infected human macrophages THP-1 blood of patients with Crohn's disease. Lancet 2004;364(9439):
cell lines treated with infliximab (5 μg/mL) for 4 h (triangle) and 1039–44.
24 h (inverted triangle). Non-treated cells (square) were used 4. Naser SA, Schwartz D, Shafran I. Isolation of Mycobacterium
as control. Shown are the mean values (±SD) of three indepen- avium subsp paratuberculosis from breast milk of Crohn's disease
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of TNF-α was detrimental to the intracellular survival of MAP. production from macrophage: possible link between MAP and
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times of infliximab exposure to macrophages prior to the
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infection. paratuberculosis PtpA is an endogenous tyrosine phosphatase se-
In conclusion, our findings suggest that infliximab treat- creted during infection. Infect Immun 2006;74(12):6540–6.
ment results in: (i) a decrease in the antibody titer of two 8. Bach H, Ko HH, Raizman EA, Attarian R, Cho B, Biet F, et al.
mycobacterial antigens that are essential for the establish- Immunogenicity of Mycobacterium avium subsp. paratuberculosis
ment of an infection, and (ii) a decrease in the survival of proteins in Crohn's disease patients. Scand J Gastroenterol 2011;46
the bacterium within human macrophages. Both observa- (1):30–9.
tions imply that in CD patients, a suppression of TNF-α 9. Bach H, Papavinasasundaram KG, Wong D, Hmama Z, Av-Gay
leads to the activation of other immunological pathways in Y. Mycobacterium tuberculosis virulence is mediated by PtpA
macrophages, which suppresses the growth of MAP in these dephosphorylation of human vacuolar protein sorting 33B.
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patients. Our findings together with the study reported by
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Nakase et al. 6 demonstrate that the regulation of specific C, Huygen K, et al. Protein kinase G from pathogenic mycobacteria
cytokines, such as TNF-α, is critical for the survival of MAP promotes survival within macrophages. Science 2004;304(5678):
within macrophages. Based on the accumulated information 1800–4.
related to the increase of TNF-α production in infections as-
sociated with MAP, a remaining question is: why is it benefi-
cial for MAP to induce TNF-α production upon infection? Horacio Bach
More studies related to the balance of cytokines and their Division of Infectious Diseases, Department of Medicine,
link to MAP survival are necessary to understand the patho- University of British Columbia, Vancouver, BC, Canada
physiology of CD. ⁎Corresponding author.
E-mail address: horacio.bach@gmail.com.
Greg Rosenfeld
Conflict of interest Brian Bressler
Division of Gastroenterology, Department of Medicine,
None. University of British Columbia, Vancouver, BC, Canada
Acknowledgments 12 January 2012
Available online xxxx
We thank Jeffrey Helm for helpful discussions.
Please cite this article as: Bach H, et al, Treatment of Crohn's disease patients with infliximab is detrimental for the survival of Mycobacterium
avium ssp. paratuberculosis within macrophages..., Journal of Crohn's and Colitis (2012), doi:10.1016/j.crohns.2012.01.011