**3DWKRJHQHVLV**

**Chapter 1**

*Propionibacterium acnes* **as a Cause of Sarcoidosis**

Sarcoidosis is one of the best-known systemic granulomatous diseases. Despite intensive investigation, however, the etiology of sarcoidosis has remained unresolved for more than 100 years [1]. Sarcoidosis seems to result from the exposure of a genetically susceptible subject to an environmental agent, and microbial etiologies of sarcoidosis have long been considered based on the clinical similarities to infectious granulomatous diseases [2]. Several epidemio‐ logic mechanisms may underlie the association of an infective agent or agents with the etiology of sarcoidosis, including spatial, seasonal, and occupational clustering [3]. The results of the ACCESS (A Case Control Etiologic Study of Sarcoidosis) study support an association between

Mycobacterial and propionibacterial organisms are the most commonly implicated etiologic agents based on studies indicating the detection by polymerase chain reaction (PCR) of microbial DNA from these organisms in tissues from sarcoid patients around the world [5-7]. Different studies have produced considerably varying results, however, with microbial DNA detected in 0% to 80% of sarcoidosis tissues and in 0% to more than 30% of control tissues [8, 9]. The failure to detect microbial DNA from these organisms in samples from some sarcoid patients suggests other causes of sarcoidosis in those patients, whereas detection of the

Immune responses against microbial antigens from these organisms, such as ESAT-6 and KatG peptides from *Mycobacterium tuberculosis* and a recombinant trigger-factor protein from *Propionibacterium acnes*, have been examined in sarcoid patients and control subjects [10, 11]. Immune responses are frequently detected in sarcoid patients as well as in some non-sarcoid patients and healthy subjects. Latent infection by these organisms complicates the interpreta‐ tion of the results of these immunologic studies. Unless microbial antigens that cause a specific immune response found only in sarcoid patients can be used to stimulate an immune response,

> © 2013 Eishi; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

microbial DNA in some control samples suggests latent infection of the bacterium.

Yoshinobu Eishi

**1. Introduction**

http://dx.doi.org/10.5772/55073

Additional information is available at the end of the chapter

selected microbially-rich environments and sarcoidosis [4].

## *Propionibacterium acnes* **as a Cause of Sarcoidosis**

### Yoshinobu Eishi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55073

### **1. Introduction**

Sarcoidosis is one of the best-known systemic granulomatous diseases. Despite intensive investigation, however, the etiology of sarcoidosis has remained unresolved for more than 100 years [1]. Sarcoidosis seems to result from the exposure of a genetically susceptible subject to an environmental agent, and microbial etiologies of sarcoidosis have long been considered based on the clinical similarities to infectious granulomatous diseases [2]. Several epidemio‐ logic mechanisms may underlie the association of an infective agent or agents with the etiology of sarcoidosis, including spatial, seasonal, and occupational clustering [3]. The results of the ACCESS (A Case Control Etiologic Study of Sarcoidosis) study support an association between selected microbially-rich environments and sarcoidosis [4].

Mycobacterial and propionibacterial organisms are the most commonly implicated etiologic agents based on studies indicating the detection by polymerase chain reaction (PCR) of microbial DNA from these organisms in tissues from sarcoid patients around the world [5-7]. Different studies have produced considerably varying results, however, with microbial DNA detected in 0% to 80% of sarcoidosis tissues and in 0% to more than 30% of control tissues [8, 9]. The failure to detect microbial DNA from these organisms in samples from some sarcoid patients suggests other causes of sarcoidosis in those patients, whereas detection of the microbial DNA in some control samples suggests latent infection of the bacterium.

Immune responses against microbial antigens from these organisms, such as ESAT-6 and KatG peptides from *Mycobacterium tuberculosis* and a recombinant trigger-factor protein from *Propionibacterium acnes*, have been examined in sarcoid patients and control subjects [10, 11]. Immune responses are frequently detected in sarcoid patients as well as in some non-sarcoid patients and healthy subjects. Latent infection by these organisms complicates the interpreta‐ tion of the results of these immunologic studies. Unless microbial antigens that cause a specific immune response found only in sarcoid patients can be used to stimulate an immune response,

© 2013 Eishi; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

immunologic approaches will not be sufficient to unequivocally confirm that these organisms are causative.

**2.2. Bacterial culture from tissue samples without sarcoidosis**

Ishige *et al* cultured peripheral lung tissue and various lymph nodes obtained from patients with diseases other than sarcoidosis [17]. *P. acnes* was isolated from 24 of 43 lungs and 8 of 11 mediastinal lymph nodes, mostly in pure culture. *P. acnes* was isolated from 10 of 20 gastric and 3 of 12 intestinal lymph nodes; intestinal bacteria were also numerous. *P. acnes* was generally the only species isolated from these tissues. The number of *P. acnes* cells isolated was usually no more than 500 colony forming units (CFU)/g in the lungs and lymph nodes. Of 43 lungs from patients without sarcoidosis, only 4 (9%) had exceptionally high numbers of *P. acnes* cells. Random amplified polymorphic DNA analysis was used to compare the DNA of 45 isolates of *P. acnes* from these patients, 39 isolates from sarcoid lymph nodes, and 67 isolates from normal skin, conjunctiva, and intestine. The *P. acnes* strains in the lung and mediastinal lymph nodes differed genetically from those in the skin. Therefore, contamination from the skin during operative or culture procedures seems unlikely. These findings suggest that *P.*

*Propionibacterium acnes* as a Cause of Sarcoidosis

http://dx.doi.org/10.5772/55073

5

*acnes* normally resides in peripheral lung tissue and mediastinal lymph nodes.

Studies of cell-invading *P. acnes* are essential for linking this indigenous bacterium to the cause of sarcoidosis because infectious granulomas are commonly caused by intracellular pathogens. Furukawa *et al* examined the cell invasiveness and serotype of *P. acnes* isolates from lymph nodes affected by sarcoidosis, together with isolates from non-sarcoid tissue obtained from the lymph nodes, lungs, prostate, skin, conjunctiva, and intestine [18]. The invasiveness of these *P. acnes* isolates into HEK293T cells was examined by cell-invasion assay according to the method described by Cue and Cleary [19] and intracellular localization of the invasive isolates was confirmed by electron microscopy (Figure 1). Cell invasiveness was found in 14 (40%) of 35 sarcoid isolates and 65 (51%) of 127 non-sarcoid isolates. The proportion of invasive isolates did not differ between isolates from sarcoid and non-sarcoid tissues. The wholebacterium enzyme-linked immunosorbent assays with serotype-specific antibodies discrimi‐ nated the serotype of all 162 isolates (112 strains of serotype I and 50 strains of serotype II). The proportion of the two serotypes did not differ between sarcoid and non-sarcoid tissues. Cell invasiveness was found in 79 (71%) of 112 serotype I isolates and in none of 50 serotype

Some investigators in Europe using PCR assays detected mycobacterial DNA in samples of affected tissue from patients with sarcoidosis [20-22], but others did not [23-25]. Quantification of the bacterial genomes detected in sarcoid lesions is essential for clarifying the etiologic correlation between lesions and bacteria detected therein because a tiny volume of bacteria or bacterial DNA can be detected even in conditions of latent infection or contamination with no

**2.3. Cell invasiveness of** *P. acnes*

**3. Polymerase chain reaction**

II isolates.

etiologic correlation.

Granuloma formation results from the persistence of a nondegradable product or a hypersen‐ sitivity response [12]. The two mechanisms overlap in most infectious diseases because microorganisms act as both foreign bodies and antigens to induce immunologic responses. Granulomas serve as protective mechanism to sequester and degrade the invading agent. The pathologic hallmark of sarcoidosis is an epithelioid cell granuloma, thus some etiologic agent of sarcoidosis must be present or have been present within the sarcoid granuloma. Histopa‐ thologic studies are therefore essential to demonstrate mycobacterial or propionibacterial organisms or antigens within sarcoid granulomas to demonstrate an etiologic link between sarcoidosis and these organisms.

*P. acnes* is so far the only microorganism isolated from sarcoid lesions by bacterial culture [13, 14]. *P. acnes* is an anaerobic, non-spore-forming, gram-positive rod bacterium indigenous to the skin and mucosal surfaces. A series of Japanese studies has provided accumulating evidence for a role of *P. acnes* in sarcoidosis. In this review, we propose mechanisms of granuloma formation in response to this indigenous bacterium in subjects with sarcoidosis based on our results obtained using histopathological and experimental approaches, and introduce a new concept of infectious disease in which endogenous infection is caused by indigenous bacteria.

### **2. Bacterial culture**

The lung and its draining lymph nodes are the organs most commonly affected by sarcoidosis. As the lung constantly encounters airborne substances, including pathogens, many researchers have considered infection to trigger sarcoidosis and have thus tried to identify possible causative transmissible agents and their contribution to the mechanism of sarcoid granuloma formation [15, 16].

#### **2.1. Bacterial culture from tissue samples affected by sarcoidosis**

In the late 1970s, a large Japanese research project conducted by many clinicians and micro‐ biologists with support by a grant from Japanese Ministry of Health was organized to seek the pathogens responsible for sarcoidosis. Extensive trials were performed to isolate microorgan‐ isms, including bacteria, viruses, and fungi, from tissue samples (especially biopsied lymph nodes) affected by sarcoidosis. Only *P. acnes,* and no other microorganism, was isolated from the large number of samples [13]. *P. acnes* was isolated in culture from biopsy samples of 31 (78%) of 40 lymph nodes from 40 patients with sarcoidosis [14], whereas this indigenous bacterium was also cultured from 20% of 141 control lymph nodes from patients with diseases other than sarcoidosis. The study was repeated twice to confirm that the initial samples had not been contaminated by cutaneous *P. acnes* during biopsy, and the results of both studies were the same.

#### **2.2. Bacterial culture from tissue samples without sarcoidosis**

Ishige *et al* cultured peripheral lung tissue and various lymph nodes obtained from patients with diseases other than sarcoidosis [17]. *P. acnes* was isolated from 24 of 43 lungs and 8 of 11 mediastinal lymph nodes, mostly in pure culture. *P. acnes* was isolated from 10 of 20 gastric and 3 of 12 intestinal lymph nodes; intestinal bacteria were also numerous. *P. acnes* was generally the only species isolated from these tissues. The number of *P. acnes* cells isolated was usually no more than 500 colony forming units (CFU)/g in the lungs and lymph nodes. Of 43 lungs from patients without sarcoidosis, only 4 (9%) had exceptionally high numbers of *P. acnes* cells. Random amplified polymorphic DNA analysis was used to compare the DNA of 45 isolates of *P. acnes* from these patients, 39 isolates from sarcoid lymph nodes, and 67 isolates from normal skin, conjunctiva, and intestine. The *P. acnes* strains in the lung and mediastinal lymph nodes differed genetically from those in the skin. Therefore, contamination from the skin during operative or culture procedures seems unlikely. These findings suggest that *P. acnes* normally resides in peripheral lung tissue and mediastinal lymph nodes.

#### **2.3. Cell invasiveness of** *P. acnes*

immunologic approaches will not be sufficient to unequivocally confirm that these organisms

Granuloma formation results from the persistence of a nondegradable product or a hypersen‐ sitivity response [12]. The two mechanisms overlap in most infectious diseases because microorganisms act as both foreign bodies and antigens to induce immunologic responses. Granulomas serve as protective mechanism to sequester and degrade the invading agent. The pathologic hallmark of sarcoidosis is an epithelioid cell granuloma, thus some etiologic agent of sarcoidosis must be present or have been present within the sarcoid granuloma. Histopa‐ thologic studies are therefore essential to demonstrate mycobacterial or propionibacterial organisms or antigens within sarcoid granulomas to demonstrate an etiologic link between

*P. acnes* is so far the only microorganism isolated from sarcoid lesions by bacterial culture [13, 14]. *P. acnes* is an anaerobic, non-spore-forming, gram-positive rod bacterium indigenous to the skin and mucosal surfaces. A series of Japanese studies has provided accumulating evidence for a role of *P. acnes* in sarcoidosis. In this review, we propose mechanisms of granuloma formation in response to this indigenous bacterium in subjects with sarcoidosis based on our results obtained using histopathological and experimental approaches, and introduce a new concept of infectious disease in which endogenous infection is caused by

The lung and its draining lymph nodes are the organs most commonly affected by sarcoidosis. As the lung constantly encounters airborne substances, including pathogens, many researchers have considered infection to trigger sarcoidosis and have thus tried to identify possible causative transmissible agents and their contribution to the mechanism of sarcoid granuloma

In the late 1970s, a large Japanese research project conducted by many clinicians and micro‐ biologists with support by a grant from Japanese Ministry of Health was organized to seek the pathogens responsible for sarcoidosis. Extensive trials were performed to isolate microorgan‐ isms, including bacteria, viruses, and fungi, from tissue samples (especially biopsied lymph nodes) affected by sarcoidosis. Only *P. acnes,* and no other microorganism, was isolated from the large number of samples [13]. *P. acnes* was isolated in culture from biopsy samples of 31 (78%) of 40 lymph nodes from 40 patients with sarcoidosis [14], whereas this indigenous bacterium was also cultured from 20% of 141 control lymph nodes from patients with diseases other than sarcoidosis. The study was repeated twice to confirm that the initial samples had not been contaminated by cutaneous *P. acnes* during biopsy, and the results of both studies

**2.1. Bacterial culture from tissue samples affected by sarcoidosis**

are causative.

4 Sarcoidosis

sarcoidosis and these organisms.

indigenous bacteria.

**2. Bacterial culture**

formation [15, 16].

were the same.

Studies of cell-invading *P. acnes* are essential for linking this indigenous bacterium to the cause of sarcoidosis because infectious granulomas are commonly caused by intracellular pathogens. Furukawa *et al* examined the cell invasiveness and serotype of *P. acnes* isolates from lymph nodes affected by sarcoidosis, together with isolates from non-sarcoid tissue obtained from the lymph nodes, lungs, prostate, skin, conjunctiva, and intestine [18]. The invasiveness of these *P. acnes* isolates into HEK293T cells was examined by cell-invasion assay according to the method described by Cue and Cleary [19] and intracellular localization of the invasive isolates was confirmed by electron microscopy (Figure 1). Cell invasiveness was found in 14 (40%) of 35 sarcoid isolates and 65 (51%) of 127 non-sarcoid isolates. The proportion of invasive isolates did not differ between isolates from sarcoid and non-sarcoid tissues. The wholebacterium enzyme-linked immunosorbent assays with serotype-specific antibodies discrimi‐ nated the serotype of all 162 isolates (112 strains of serotype I and 50 strains of serotype II). The proportion of the two serotypes did not differ between sarcoid and non-sarcoid tissues. Cell invasiveness was found in 79 (71%) of 112 serotype I isolates and in none of 50 serotype II isolates.

### **3. Polymerase chain reaction**

Some investigators in Europe using PCR assays detected mycobacterial DNA in samples of affected tissue from patients with sarcoidosis [20-22], but others did not [23-25]. Quantification of the bacterial genomes detected in sarcoid lesions is essential for clarifying the etiologic correlation between lesions and bacteria detected therein because a tiny volume of bacteria or bacterial DNA can be detected even in conditions of latent infection or contamination with no etiologic correlation.

**Figure 1.** Invasiveness of *P. acnes* into epithelial cells. HEK293T cells infected with one of the serotype 1 *P. acnes* strains isolated from sarcoid lymph nodes were Giemsa-stained (left) and further examined by electron microscopy (right). The electron micrographs of the cells infected with an invasive isolate show intracellular localization of the bacterium (indicated by the red arrows).

#### **3.1. Quantitative PCR for propionibacterial and mycobacterial DNA**

Ishige *et al* used quantitative PCR to search for bacterial genomes of *P. acnes*, *P. granulosum*, and *M. tuberculosis* in histologic sections of lymph nodes from patients with sarcoidosis, tuberculosis, or gastric cancer [26]. They examined lymph node biopsy samples from 15 patients with sarcoidosis and 15 patients with tuberculosis lymphadenitis. As controls, they examined 15 lymph nodes without metastasis from 15 patients with gastric cancer undergoing surgery (Figure 2). Genomes of *M. tuberculosis* were found in samples from all 15 patients with tuberculosis, 3 patients with sarcoidosis, and 1 control sample. Genomes of *P. acnes* were found in 12 of 15 patients with sarcoidosis, 2 tuberculosis patients, and 3 controls. The difference in the estimated number of *P. acnes* genomes between individuals with and without sarcoidosis was similar to that in the number of *M. tuberculosis* between people with and without tuber‐ culosis. Biopsy samples from the three patients with sarcoidosis but without *P. acnes* all contained many *P. granulosum* DNA. These findings suggest that propionibacteria resided in or proliferated ectopically in the sarcoid lesions, whether or not there was a connection with the disease. Propionibacteria are more likely than mycobacteria to cause sarcoidosis.

two of the sarcoid samples. *M. avium* subsp. *paratuberculosis* was not found in any sarcoid sample. *M. tuberculosis* was found in only 0% to 9% of the sarcoid samples, but in 65% to 100% of the tuberculosis samples. In sarcoid lymph nodes, the total numbers of genomes of *P. acnes* or *P. granulosum* far exceeded those of *M. tuberculosis*. *P. acnes* or *P. granulosum* was found in 0% to 60% of the tuberculosis and control samples, but the total numbers of genomes of *P. acnes* or *P. granulosum* in these samples were lower than those found in sarcoid samples. *Propionibacteria* spp. are more likely than *Mycobacteria* spp. to be involved in the etiology of

**Figure 2.** Quantitative PCR of bacterial DNA in lymph nodes from patients with sarcoidosis, tuberculosis, and gastric cancer. The horizontal dotted lines show the detection threshold and samples with results under this line were consid‐

Detection threshold

http://dx.doi.org/10.5772/55073

7

**Cont**

●●● ● ●● ●● ● ●● ●● ● ●

● ●

10

2.5

●●● ●● ●●●●● ●● ● ● ●

●

●

●

● ● ●

●

●

●

● ●● ● ● ● ● ● ● ● ● ● ●● ●

●

**Sar Tbc**

■

*Propionibacterium acnes* as a Cause of Sarcoidosis

■

■

100

1000

10000

100000

*In situ* localization of *P. acnes* genomes in sarcoid lymph nodes may help to elucidate an etiologic link between sarcoidosis and this indigenous bacterium. Formalin-fixed and paraffinembedded biopsy samples of lymph nodes from nine patients with sarcoidosis, nine patients with tuberculosis, and nine patients with nonspecific lymphadenitis as controls were examined by quantitative real-time PCR (QPCR) for *P. acnes* and by *in situ* hybridization (ISH) using catalyzed reporter deposition (CARD) for signal amplification with digoxigenin-labeled oligonucleotide probes that complemented 16S rRNA of *P. acnes* [27]. The signals per 250

of tissue sections from inside and outside sarcoidosis and tuberculosis granulomas and

sarcoidosis, not only in Japanese but also in European patients with sarcoidosis.

**4.** *In situ* **hybridization**

10

2.5

ered negative.

100

Number of bacterial genomes (x102)

1000

10000

●

● ●●

●● ● ● ●

●

●

●

■■■ ●●●● ● ●● ● ● ●●● ●

●

●

**Sar Tbc Cont**

●● ● ● ● ● ● ● ● ● ●●● ● ●

10

2.5

● ● ● ● ●

●● ● ●● ●● ● ● ● ●●

● ● ● ● ●● ●

● ●

●

● Sar: Sarcoisosis ● Tbc: Tuberculosis ● Cont: Control

●

**Sar Tbc Cont**

*P. acnes M. tuberculosis P. granulosum* 

●

●

100

1000

10000

100000

100000

µm2

#### **3.2. International collaborative study with quantitative real-time PCR**

The international collaborative study evaluated the possible etiologic link between sarcoidosis and the suspected bacterial species [8]. Formalin-fixed and paraffin-embedded sections of biopsy samples of lymph nodes, 1 from each of 108 patients with sarcoidosis and 65 patients with tuberculosis, together with 86 control samples, were collected from 2 institutes in Japan and 3 institutes in Italy, Germany, and England (Figure 3). Genomes of *P. acnes*, *P. granulo‐ sum*, *M. tuberculosis*, *M. avium* subsp. *paratuberculosis*, and *Escherichia coli* (as the control) were estimated by quantitative real-time PCR. Either *P. acnes* or *P. granulosum* was found in all but

**Figure 2.** Quantitative PCR of bacterial DNA in lymph nodes from patients with sarcoidosis, tuberculosis, and gastric cancer. The horizontal dotted lines show the detection threshold and samples with results under this line were consid‐ ered negative.

two of the sarcoid samples. *M. avium* subsp. *paratuberculosis* was not found in any sarcoid sample. *M. tuberculosis* was found in only 0% to 9% of the sarcoid samples, but in 65% to 100% of the tuberculosis samples. In sarcoid lymph nodes, the total numbers of genomes of *P. acnes* or *P. granulosum* far exceeded those of *M. tuberculosis*. *P. acnes* or *P. granulosum* was found in 0% to 60% of the tuberculosis and control samples, but the total numbers of genomes of *P. acnes* or *P. granulosum* in these samples were lower than those found in sarcoid samples. *Propionibacteria* spp. are more likely than *Mycobacteria* spp. to be involved in the etiology of sarcoidosis, not only in Japanese but also in European patients with sarcoidosis.

### **4.** *In situ* **hybridization**

**3.1. Quantitative PCR for propionibacterial and mycobacterial DNA**

(indicated by the red arrows).

6 Sarcoidosis

Ishige *et al* used quantitative PCR to search for bacterial genomes of *P. acnes*, *P. granulosum*, and *M. tuberculosis* in histologic sections of lymph nodes from patients with sarcoidosis, tuberculosis, or gastric cancer [26]. They examined lymph node biopsy samples from 15 patients with sarcoidosis and 15 patients with tuberculosis lymphadenitis. As controls, they examined 15 lymph nodes without metastasis from 15 patients with gastric cancer undergoing surgery (Figure 2). Genomes of *M. tuberculosis* were found in samples from all 15 patients with tuberculosis, 3 patients with sarcoidosis, and 1 control sample. Genomes of *P. acnes* were found in 12 of 15 patients with sarcoidosis, 2 tuberculosis patients, and 3 controls. The difference in the estimated number of *P. acnes* genomes between individuals with and without sarcoidosis was similar to that in the number of *M. tuberculosis* between people with and without tuber‐ culosis. Biopsy samples from the three patients with sarcoidosis but without *P. acnes* all contained many *P. granulosum* DNA. These findings suggest that propionibacteria resided in or proliferated ectopically in the sarcoid lesions, whether or not there was a connection with

**Figure 1.** Invasiveness of *P. acnes* into epithelial cells. HEK293T cells infected with one of the serotype 1 *P. acnes* strains isolated from sarcoid lymph nodes were Giemsa-stained (left) and further examined by electron microscopy (right). The electron micrographs of the cells infected with an invasive isolate show intracellular localization of the bacterium

the disease. Propionibacteria are more likely than mycobacteria to cause sarcoidosis.

The international collaborative study evaluated the possible etiologic link between sarcoidosis and the suspected bacterial species [8]. Formalin-fixed and paraffin-embedded sections of biopsy samples of lymph nodes, 1 from each of 108 patients with sarcoidosis and 65 patients with tuberculosis, together with 86 control samples, were collected from 2 institutes in Japan and 3 institutes in Italy, Germany, and England (Figure 3). Genomes of *P. acnes*, *P. granulo‐ sum*, *M. tuberculosis*, *M. avium* subsp. *paratuberculosis*, and *Escherichia coli* (as the control) were estimated by quantitative real-time PCR. Either *P. acnes* or *P. granulosum* was found in all but

**3.2. International collaborative study with quantitative real-time PCR**

*In situ* localization of *P. acnes* genomes in sarcoid lymph nodes may help to elucidate an etiologic link between sarcoidosis and this indigenous bacterium. Formalin-fixed and paraffinembedded biopsy samples of lymph nodes from nine patients with sarcoidosis, nine patients with tuberculosis, and nine patients with nonspecific lymphadenitis as controls were examined by quantitative real-time PCR (QPCR) for *P. acnes* and by *in situ* hybridization (ISH) using catalyzed reporter deposition (CARD) for signal amplification with digoxigenin-labeled oligonucleotide probes that complemented 16S rRNA of *P. acnes* [27]. The signals per 250 µm2 of tissue sections from inside and outside sarcoidosis and tuberculosis granulomas and Figure 3

**Figure 3.** Quantitative real-time PCR of bacterial DNA in lymph node samples from Japanese and European patients with sarcoidosis. The horizontal red lines show the detection threshold and samples with results under this line were considered negative.

from control lymph nodes were counted. The number of genomes determined by QPCR was examined for correlation with the mean signal count by ISH with CARD. In sarcoid samples, one or several signals were detected in the cytoplasm of some epithelioid cells in granulomas (Figure 4). The mean signal counts were higher in granulomatous areas than in other areas of sarcoid lymph nodes. The correlation between the QPCR and ISH with CARD results was significant (*r* = 0.86, *p* < 0.001). The accumulation of *P. acnes* genomes in and around sarcoid granulomas suggests that this indigenous bacterium is related to the cause of granulomatous inflammation in sarcoidosis.

(PAB antibody) and ribosome-bound trigger factor protein (TIG antibody). They examined formalin-fixed and paraffin-embedded samples of lungs and lymph nodes from 196 patients with sarcoidosis, and corresponding control samples from 275 patients with non-sarcoidosis diseases. The samples were mostly from Japanese patients, with 64 lymph node samples from

**Figure 4.** *In situ* hybridization using catalyzed reporter deposition for signal amplification with digoxigenin-labeled oligonucleotide probes that complemented 16S rRNA of *P. acnes*. Many signals were detected in the cytoplasm of sar‐

*Propionibacterium acnes* as a Cause of Sarcoidosis

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9

Immunohistochemistry with the PAB antibody revealed small round bodies within sarcoid granulomas in 20/27 (74%) video-assisted thoracic surgery lung samples, 24/50 (48%) trans‐ bronchial lung biopsy samples, 71/81 (88%) Japanese lymph node samples, and 34/38 (89%) German lymph node samples. The PAB antibody did not react with non-sarcoid granulomas in any of the 45 tuberculosis samples or the 34 samples with sarcoid reaction. The appearance of the small round bodies detected by the PAB antibody within sarcoid granulomas did not differ between lungs and lymph nodes. In sarcoid granulomas with many small round bodies, the cytoplasm of some granuloma cells was filled with small round bodies, consistent with the intracellular proliferation of the bacterium (Figure 5). In many sarcoid granulomas, a few small round bodies with occasional degraded or large-sized features were scattered among the granuloma cells. The amount of these small round bodies varied from each granuloma in identical sarcoid samples as well as from each sarcoid tissue sample (Figure 6). The appearance of the small round bodies detected by the PAB antibody within sarcoid granulomas did not

**5.1. Intracellular** *P. acnes* **detected within sarcoid granuloma**

differ between lungs and lymph nodes (Figure 7, 8).

German patients [28].

coid granuloma cells.

#### **5. Immunohistochemistry**

Granulomatous reactions are basically a defense mechanism that the body uses to fight off poorly degradable antigens. Granulomas begin as a small collection of lymphocytes and macrophages surrounding poorly degradable antigens. The aggregating macrophages, called an early focus of granuloma, then change to epithelioid cells and become organized into a cluster of cells, called an immature granuloma. Further progression results in ball-like clusters of cells and fusion of macrophages into giant cells, called a mature granuloma. The questions that must be asked in searching for the cause of sarcoidosis, therefore, are: "What is the antigen that the granulomas are fighting?" and "How is the antigen localized within the sarcoid lesion?" To evaluate the pathogenic role of *P. acnes*, Negi *et al* screened for this indigenous bacterium in sarcoid and non-sarcoid tissues using immunohistochemical methods with novel *P. acnes*-specific monoclonal antibodies that react with cell-membrane-bound lipoteichoic acid

**Figure 4.** *In situ* hybridization using catalyzed reporter deposition for signal amplification with digoxigenin-labeled oligonucleotide probes that complemented 16S rRNA of *P. acnes*. Many signals were detected in the cytoplasm of sar‐ coid granuloma cells.

(PAB antibody) and ribosome-bound trigger factor protein (TIG antibody). They examined formalin-fixed and paraffin-embedded samples of lungs and lymph nodes from 196 patients with sarcoidosis, and corresponding control samples from 275 patients with non-sarcoidosis diseases. The samples were mostly from Japanese patients, with 64 lymph node samples from German patients [28].

#### **5.1. Intracellular** *P. acnes* **detected within sarcoid granuloma**

from control lymph nodes were counted. The number of genomes determined by QPCR was examined for correlation with the mean signal count by ISH with CARD. In sarcoid samples, one or several signals were detected in the cytoplasm of some epithelioid cells in granulomas (Figure 4). The mean signal counts were higher in granulomatous areas than in other areas of sarcoid lymph nodes. The correlation between the QPCR and ISH with CARD results was significant (*r* = 0.86, *p* < 0.001). The accumulation of *P. acnes* genomes in and around sarcoid granulomas suggests that this indigenous bacterium is related to the cause of granulomatous

**Figure 3.** Quantitative real-time PCR of bacterial DNA in lymph node samples from Japanese and European patients with sarcoidosis. The horizontal red lines show the detection threshold and samples with results under this line were

**PA PG TB EC PA PG TB EC PA PG TB EC PA PG TB EC PA PG TB EC**

PA: *P. acnes* PG: *P. granulosum* TB: *M. tuberculosis* EC*: E. coli* 

**2/17 0/17**

**27/33 12/33**

**16/17**

**14/17**

Italy (n=17) Germany (n=33)

England (n=15)

**10/15**

**0/15 1/15**

**0**

**200**

**2/33 1/33** **400**

**600**

**800**

**1000**

**1200**

**1400**

**15/15**

Granulomatous reactions are basically a defense mechanism that the body uses to fight off poorly degradable antigens. Granulomas begin as a small collection of lymphocytes and macrophages surrounding poorly degradable antigens. The aggregating macrophages, called an early focus of granuloma, then change to epithelioid cells and become organized into a cluster of cells, called an immature granuloma. Further progression results in ball-like clusters of cells and fusion of macrophages into giant cells, called a mature granuloma. The questions that must be asked in searching for the cause of sarcoidosis, therefore, are: "What is the antigen that the granulomas are fighting?" and "How is the antigen localized within the sarcoid lesion?" To evaluate the pathogenic role of *P. acnes*, Negi *et al* screened for this indigenous bacterium in sarcoid and non-sarcoid tissues using immunohistochemical methods with novel *P. acnes*-specific monoclonal antibodies that react with cell-membrane-bound lipoteichoic acid

inflammation in sarcoidosis.

Tokyo, Japan (n=24)

Figure 3

**11/24**

considered negative.

**Genomes/500ng extracted DNA**

8 Sarcoidosis

Kumamoto, Japan (n=19)

**12/19**

**50 50 50 50**

**0/19 1/19**

**5. Immunohistochemistry**

**0**

**200** 

**0/24 0/24**

**400** 

**600** 

**800** 

**1000** 

**1200** 

**1400** 

**19/24 16/19**

Immunohistochemistry with the PAB antibody revealed small round bodies within sarcoid granulomas in 20/27 (74%) video-assisted thoracic surgery lung samples, 24/50 (48%) trans‐ bronchial lung biopsy samples, 71/81 (88%) Japanese lymph node samples, and 34/38 (89%) German lymph node samples. The PAB antibody did not react with non-sarcoid granulomas in any of the 45 tuberculosis samples or the 34 samples with sarcoid reaction. The appearance of the small round bodies detected by the PAB antibody within sarcoid granulomas did not differ between lungs and lymph nodes. In sarcoid granulomas with many small round bodies, the cytoplasm of some granuloma cells was filled with small round bodies, consistent with the intracellular proliferation of the bacterium (Figure 5). In many sarcoid granulomas, a few small round bodies with occasional degraded or large-sized features were scattered among the granuloma cells. The amount of these small round bodies varied from each granuloma in identical sarcoid samples as well as from each sarcoid tissue sample (Figure 6). The appearance of the small round bodies detected by the PAB antibody within sarcoid granulomas did not differ between lungs and lymph nodes (Figure 7, 8).

**Figure 5.** Immunohistochemistry with a *P. acnes*-specific monoclonal antibody (PAB antibody) that reacts with cellmembrane-bound lipoteichoic acid of the bacterium. Many small round bodies are shown within a non-caseating epi‐ thelioid cell granuloma of sarcoid lymph node.

**Figure 7.** In the lung sarcoid granuloma lesion surrounded by prominent inflammatory cell infiltration, small round bodies are detected by the PAB antibody not only in the granuloma cells but also in some of the inflammatory cells.

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**Figure 8.** Higher magnification of the area indicated by the arrow in Figure 7. Some swollen macrophages of the im‐

mature granuloma are filled with many small round bodies detected by the PAB antibody.

The arrow indicates the magnified region shown in Fig. 8.

**Figure 6.** Many small round bodies detected by the PAB antibody are shown intermingled with many lymphocytes in one immature granuloma (right), but only a few are observed in the mature granuloma (left) of the sarcoid lymph node. Most of the *P. acnes* are present within the granuloma, but some are present outside of the granuloma (as indi‐ cated by the arrow).

**Figure 7.** In the lung sarcoid granuloma lesion surrounded by prominent inflammatory cell infiltration, small round bodies are detected by the PAB antibody not only in the granuloma cells but also in some of the inflammatory cells. The arrow indicates the magnified region shown in Fig. 8.

**Figure 5.** Immunohistochemistry with a *P. acnes*-specific monoclonal antibody (PAB antibody) that reacts with cellmembrane-bound lipoteichoic acid of the bacterium. Many small round bodies are shown within a non-caseating epi‐

**Figure 6.** Many small round bodies detected by the PAB antibody are shown intermingled with many lymphocytes in one immature granuloma (right), but only a few are observed in the mature granuloma (left) of the sarcoid lymph node. Most of the *P. acnes* are present within the granuloma, but some are present outside of the granuloma (as indi‐

thelioid cell granuloma of sarcoid lymph node.

10 Sarcoidosis

cated by the arrow).

**Figure 8.** Higher magnification of the area indicated by the arrow in Figure 7. Some swollen macrophages of the im‐ mature granuloma are filled with many small round bodies detected by the PAB antibody.

#### **5.2. Intracellular** *P. acnes* **in non-granulomatous areas**

In non-granulomatous areas, small round bodies detected by the PAB antibody were found in alveolar macrophages of lungs and paracortical macrophages of lymph nodes from many sarcoid and some non-sarcoid patients. In the lymph nodes, paracortical macrophages with many small round bodies detected by the PAB antibody (Figure 9) were observed in 26 (22%) of 119 sarcoid samples and 18 (11%) of 165 non-sarcoid samples. The frequency was signifi‐ cantly higher in the sarcoid samples. Such small round bodies were observed in lymphatic endothelial cells in a few samples of sarcoid lymph nodes (Figure 10). In the lungs, alveolar macrophages with many small round bodies detected by the PAB antibody were found in 28 (36%) of 77 sarcoid samples and 18 (16%) of 110 non-sarcoid samples. The frequency was significantly higher in sarcoid samples. Such alveolar macrophages occasionally contained one or a few large spheroidal bodies detected by the PAB antibody that were acid-fast with Fite staining and also reacted with the TIG antibody.

antibody and TIG antibody were differentially distributed in the outer and inner areas of the HW bodies, respectively (Figure 12). The localization of cell-membrane-bound lipoteichoic acid detected by the PAB antibody and ribosome-bound trigger factor detected by the TIG antibody suggests that HW might not be phagolysosomally-degraded products of *P. acnes*, but rather intact forms of intracellular bacteria because the original distribution pattern (plasma‐ lemmal and protoplasmic localization, respectively) of these bacterial components was preserved in terms of the morphologic structure of the bacterium. Furthermore, conventional electron microscopy revealed that these bodies lack a cell-wall structure and occasionally exhibit protrusions from the body that appear to be yeast-like proliferating features (not mitotic, but sprouting or branching), characteristic of cell-wall-deficient (L-form) bacteria

**Figure 10.** Some of the small round bodies detected by the PAB antibody (green arrows) were observed in lymphatic

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Histopathologic analysis with the PAB antibody led us to formulate a hypothesis for the mechanism of granuloma formation in sarcoidosis (Figure 13). *P. acnes* causes latent infection and persists in macrophages. HW bodies are dormant and cell-wall-deficient *P. acnes*. This dormant form of *P. acnes* can be activated endogenously under certain environmental condi‐ tions and proliferate in cells at the sites of latent infection. Small round bodies proliferating in macrophages are infective forms of *P. acnes*. When these bodies spread out of macrophages, they infect other cells or organs via the lymph and blood streams. Sarcoid granulomas are formed as a host defense mechanism at the sites of activated bacteria proliferating intracellu‐ larly in patients with hypersensitive immune responses to *P. acnes* to prevent the spread of the

(Figure 9). HW bodies may be cell-wall-deficient *P. acnes*.

endothelial cells adjacent to sarcoid granulomas of the lymph node.

**5.4. Intracellular proliferation of** *P. acnes*

infectious agent.

**Figure 9.** A cluster of some swollen macrophages filled with many small round bodies detected by the PAB antibody is occasionally found in paracortical areas of sarcoid lymph nodes. The arrow indicates a large-spheroidal body similar to Hamazaki-Wesenberg bodies.

#### **5.3. Hamazaki-Wesenberg bodies**

Hamazaki-Wesenberg (HW) bodies frequently appear in sarcoid lymph nodes although these bodies are not specific to sarcoidosis [29-31]. The large-spheroidal acid-fast bodies, HW bodies, which were found in 50% of sarcoid and 15% of non-sarcoid lymph node samples, reacted with both PAB and TIG antibodies. Electron microscopy revealed that these HW bodies had a single bacterial structure and lacked a cell wall with occasional protrusions from the body (Figure 11). Immunoelectron microscopy revealed that the immunoreactive products of the PAB

**Figure 10.** Some of the small round bodies detected by the PAB antibody (green arrows) were observed in lymphatic endothelial cells adjacent to sarcoid granulomas of the lymph node.

antibody and TIG antibody were differentially distributed in the outer and inner areas of the HW bodies, respectively (Figure 12). The localization of cell-membrane-bound lipoteichoic acid detected by the PAB antibody and ribosome-bound trigger factor detected by the TIG antibody suggests that HW might not be phagolysosomally-degraded products of *P. acnes*, but rather intact forms of intracellular bacteria because the original distribution pattern (plasma‐ lemmal and protoplasmic localization, respectively) of these bacterial components was preserved in terms of the morphologic structure of the bacterium. Furthermore, conventional electron microscopy revealed that these bodies lack a cell-wall structure and occasionally exhibit protrusions from the body that appear to be yeast-like proliferating features (not mitotic, but sprouting or branching), characteristic of cell-wall-deficient (L-form) bacteria (Figure 9). HW bodies may be cell-wall-deficient *P. acnes*.

#### **5.4. Intracellular proliferation of** *P. acnes*

**5.2. Intracellular** *P. acnes* **in non-granulomatous areas**

12 Sarcoidosis

staining and also reacted with the TIG antibody.

Hamazaki-Wesenberg bodies.

**5.3. Hamazaki-Wesenberg bodies**

In non-granulomatous areas, small round bodies detected by the PAB antibody were found in alveolar macrophages of lungs and paracortical macrophages of lymph nodes from many sarcoid and some non-sarcoid patients. In the lymph nodes, paracortical macrophages with many small round bodies detected by the PAB antibody (Figure 9) were observed in 26 (22%) of 119 sarcoid samples and 18 (11%) of 165 non-sarcoid samples. The frequency was signifi‐ cantly higher in the sarcoid samples. Such small round bodies were observed in lymphatic endothelial cells in a few samples of sarcoid lymph nodes (Figure 10). In the lungs, alveolar macrophages with many small round bodies detected by the PAB antibody were found in 28 (36%) of 77 sarcoid samples and 18 (16%) of 110 non-sarcoid samples. The frequency was significantly higher in sarcoid samples. Such alveolar macrophages occasionally contained one or a few large spheroidal bodies detected by the PAB antibody that were acid-fast with Fite

**Figure 9.** A cluster of some swollen macrophages filled with many small round bodies detected by the PAB antibody is occasionally found in paracortical areas of sarcoid lymph nodes. The arrow indicates a large-spheroidal body similar to

Hamazaki-Wesenberg (HW) bodies frequently appear in sarcoid lymph nodes although these bodies are not specific to sarcoidosis [29-31]. The large-spheroidal acid-fast bodies, HW bodies, which were found in 50% of sarcoid and 15% of non-sarcoid lymph node samples, reacted with both PAB and TIG antibodies. Electron microscopy revealed that these HW bodies had a single bacterial structure and lacked a cell wall with occasional protrusions from the body (Figure 11). Immunoelectron microscopy revealed that the immunoreactive products of the PAB

Histopathologic analysis with the PAB antibody led us to formulate a hypothesis for the mechanism of granuloma formation in sarcoidosis (Figure 13). *P. acnes* causes latent infection and persists in macrophages. HW bodies are dormant and cell-wall-deficient *P. acnes*. This dormant form of *P. acnes* can be activated endogenously under certain environmental condi‐ tions and proliferate in cells at the sites of latent infection. Small round bodies proliferating in macrophages are infective forms of *P. acnes*. When these bodies spread out of macrophages, they infect other cells or organs via the lymph and blood streams. Sarcoid granulomas are formed as a host defense mechanism at the sites of activated bacteria proliferating intracellu‐ larly in patients with hypersensitive immune responses to *P. acnes* to prevent the spread of the infectious agent.

sarcoid granulomas of the lymph node.

**5.3. Hamazaki-Wesenberg bodies** 

Dormant (latent)

**6. Host factor**

activation Th1 response

Infective (activated)

**Figure 13.** Hypothesized mechanism of sarcoid granuloma formation caused by *P. acnes*. Intracellular proliferation of *P. acnes* in macrophages triggers granuloma formation in patients with hypersensitivity to this indigenous bacterium.

The PAB antibody seems to be appropriate for detecting cell-wall-deficient *P. acnes* because an epitope of lipoteichoic acid detected by this antibody is more exposed in cell-wall-deficient forms than in conventional forms of the bacterium. The high frequency and specificity of *P. acnes* detected by the PAB antibody within sarcoid granulomas suggests an etiologic link between sarcoidosis and this indigenous bacterium. The PAB antibody may be useful for diagnosing sarcoidosis caused by *P. acnes*, when the reactivity is detected in idiopathic granulomas (Figures 14, 15). The TIG antibody seems to be appropriate for detecting latent forms of *P. acnes* because increased expression of the trigger-factor protein is found only in the HW bodies. The trigger-factor protein is a molecular chaperone, like some heat-shock proteins, and either overproduction or depletion of the trigger-factor protein causes filamentation indicative of cell division defects. Increased expression of the trigger-factor protein in HW bodies might be necessary to sustain the latent phase of intracellular persistent bacterium.

Host factors may be more critical than agent factors in the etiology of sarcoidosis, as suggested by the Kveim test phenomenon [32], in which an intracutaneously injected suspension of sarcoid tissue causes sarcoid granulomas in patients with sarcoidosis but not in healthy people or patients with other diseases. The inflammatory response in sarcoidosis involves many activated T cells and macrophages [33], with a pattern of cytokine production in the lungs

Granuloma formation

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small round bodies

**5.5. Histopathological diagnosis of sarcoidosis by the PAB antibody**

HW body Environmental factor Host factor

Figure 10.Some of the small round bodies detected by the PAB antibody (green arrows) were observed in lymphatic endothelial cells adjacent to

Hamazaki-Wesenberg (HW) bodies frequently appear in sarcoid lymph nodes although these bodies are not specific to sarcoidosis [29-31]. The large-spheroidal acid-fast bodies, HW bodies, which were found in 50% of sarcoid and 15% of non-sarcoid lymph node samples, reacted with both PAB and TIG antibodies. Electron microscopy revealed that these HW bodies had a single bacterial structure and lacked a cell wall with occasional protrusions from the body (Figure 11). Immunoelectron microscopy revealed that the immunoreactive products of the PAB antibody and TIG antibody were differentially distributed in the outer and inner areas of the HW bodies, respectively (Figure 12). The localization of cell-membrane-bound lipoteichoic acid detected by the PAB antibody and ribosome-bound trigger factor detected by the TIG antibody suggests that HW might not be phagolysosomally-degraded

bacterium. Furthermore, conventional electron microscopy revealed that these bodies lack a cell-wall structure and occasionally exhibit protrusions from the body that appear to be yeast-like proliferating features (not mitotic, but sprouting or branching),

characteristic of cell-wall-deficient (L-form) bacteria (Figure 9). HW bodies may be cell-wall-deficient *P. acnes*.

indicated by the green arrow, with hematoxylin and eosin staining (a). These bodies are strongly acid-fast with Fite staining (b). HW bodies with one-by-one protrusions (c), as indicated by black arrows, are rarely found in sinus macrophages of sarcoid lymph nodes. **Figure 11.** Hamazaki-Wesenberg (HW) bodies in sarcoid lymph nodes. HW bodies are large and spheroidal in shape with a yellow-brown color, as indicated by the green arrow, with hematoxylin and eosin staining (a). These bodies are strongly acid-fast with Fite staining (b). HW bodies with one-by-one protrusions (c), as indicated by black arrows, are rarely found in sinus macrophages of sarcoid lymph nodes.

Figure 11.Hamazaki-Wesenberg (HW) bodies in sarcoid lymph nodes. HW bodies are large and spheroidal in shape with a yellow-brown color, as

**Figure 13.** Hypothesized mechanism of sarcoid granuloma formation caused by *P. acnes*. Intracellular proliferation of *P. acnes* in macrophages triggers granuloma formation in patients with hypersensitivity to this indigenous bacterium.

#### **5.5. Histopathological diagnosis of sarcoidosis by the PAB antibody**

The PAB antibody seems to be appropriate for detecting cell-wall-deficient *P. acnes* because an epitope of lipoteichoic acid detected by this antibody is more exposed in cell-wall-deficient forms than in conventional forms of the bacterium. The high frequency and specificity of *P. acnes* detected by the PAB antibody within sarcoid granulomas suggests an etiologic link between sarcoidosis and this indigenous bacterium. The PAB antibody may be useful for diagnosing sarcoidosis caused by *P. acnes*, when the reactivity is detected in idiopathic granulomas (Figures 14, 15). The TIG antibody seems to be appropriate for detecting latent forms of *P. acnes* because increased expression of the trigger-factor protein is found only in the HW bodies. The trigger-factor protein is a molecular chaperone, like some heat-shock proteins, and either overproduction or depletion of the trigger-factor protein causes filamentation indicative of cell division defects. Increased expression of the trigger-factor protein in HW bodies might be necessary to sustain the latent phase of intracellular persistent bacterium.

#### **6. Host factor**

Figure 10.Some of the small round bodies detected by the PAB antibody (green arrows) were observed in lymphatic endothelial cells adjacent to

Hamazaki-Wesenberg (HW) bodies frequently appear in sarcoid lymph nodes although these bodies are not specific to sarcoidosis [29-31]. The large-spheroidal acid-fast bodies, HW bodies, which were found in 50% of sarcoid and 15% of non-sarcoid lymph node samples, reacted with both PAB and TIG antibodies. Electron microscopy revealed that these HW bodies had a single bacterial structure and lacked a cell wall with occasional protrusions from the body (Figure 11). Immunoelectron microscopy revealed that the immunoreactive products of the PAB antibody and TIG antibody were differentially distributed in the outer and inner areas of the HW bodies, respectively (Figure 12). The localization of cell-membrane-bound lipoteichoic acid detected by the PAB antibody and ribosome-bound trigger factor detected by the TIG antibody suggests that HW might not be phagolysosomally-degraded products of *P. acnes*, but rather intact forms of intracellular bacteria because the original distribution pattern (plasmalemmal and protoplasmic localization, respectively) of these bacterial components was preserved in terms of the morphologic structure of the bacterium. Furthermore, conventional electron microscopy revealed that these bodies lack a cell-wall structure and occasionally exhibit protrusions from the body that appear to be yeast-like proliferating features (not mitotic, but sprouting or branching),

Figure 11.Hamazaki-Wesenberg (HW) bodies in sarcoid lymph nodes. HW bodies are large and spheroidal in shape with a yellow-brown color, as indicated by the green arrow, with hematoxylin and eosin staining (a). These bodies are strongly acid-fast with Fite staining (b). HW bodies with

**Figure 11.** Hamazaki-Wesenberg (HW) bodies in sarcoid lymph nodes. HW bodies are large and spheroidal in shape with a yellow-brown color, as indicated by the green arrow, with hematoxylin and eosin staining (a). These bodies are strongly acid-fast with Fite staining (b). HW bodies with one-by-one protrusions (c), as indicated by black arrows, are

Histopathologic analysis with the PAB antibody led us to formulate a hypothesis for the mechanism of granuloma formation in sarcoidosis (Figure 13). *P. acnes* causes latent infection and persists in macrophages. HW bodies are dormant and cell-wall-deficient

PAB antibody

TIG antibody

**Figure 12.** Immuno-electron-microscopic analysis with PAB and TIG antibodies suggests HW bodies may be cell-wall-

5μm

5μm

one-by-one protrusions (c), as indicated by black arrows, are rarely found in sinus macrophages of sarcoid lymph nodes.

**5.4. Intracellular proliferation of** *P. acnes*

deficient *P. acnes*.

L-form structure HW bodies

cell-membrane-bound lipoteichoic acid

ribosome-bound trigger factor protein

rarely found in sinus macrophages of sarcoid lymph nodes.

Figure 12.Immuno-electron-microscopic analysis with PAB and TIG antibodies suggests HW bodies may be cell-wall-deficient *P. acnes*.

characteristic of cell-wall-deficient (L-form) bacteria (Figure 9). HW bodies may be cell-wall-deficient *P. acnes*.

sarcoid granulomas of the lymph node.

14 Sarcoidosis

**5.3. Hamazaki-Wesenberg bodies** 

Host factors may be more critical than agent factors in the etiology of sarcoidosis, as suggested by the Kveim test phenomenon [32], in which an intracutaneously injected suspension of sarcoid tissue causes sarcoid granulomas in patients with sarcoidosis but not in healthy people or patients with other diseases. The inflammatory response in sarcoidosis involves many activated T cells and macrophages [33], with a pattern of cytokine production in the lungs

consistent with a helper T-cell type 1 (Th1) immune response triggered by undefined antigen(s) [34]. If a propionibacterium caused a particular case of sarcoidosis, it is likely that an antigen

Ebe and colleagues searched for propionibacterial antigens that evoked cellular immune responses only in patients with sarcoidosis [11]. For this purpose, a λgt11 genomic DNA library of *P. acnes* was screened with sera from patients with sarcoidosis, because high levels of serum antibodies against the antigen usually accompany such an immune response. Of 180,000 plaques screened, 2 clones coded for an identical recombinant protein, RP35, recognized by sera. RP35, a recombinant protein of 256 amino acid residues with a calculated molecular mass of 28,133 Da, is a fragment (the C-terminal region) of the *P. acnes* trigger factor, which has 529 amino acid residues and a calculated molecular mass of 57,614 Da. The C-terminal sequence (Asp-463 to Lys-529) seems to be unique to *P. acnes*, with no similarity to sequences of other bacterial proteins deposited in the Swiss-Prot database. Conformational analysis of the Ser-491 to Lys-529 region at the C terminus revealed it to be highly antigenic. RP35 caused sarcoidosis-specific prolifera‐ tion of peripheral blood mononuclear cells (PBMCs) from 9 (18%) of 50 patients with sarcoido‐ sis (Figure 16). The same study established that serum levels of IgG and IgA antibodies to RP35 are high in patients with sarcoidosis and other lung diseases. In bronchoalveolar lavage (BAL) fluid, IgG and IgA antibody levels were high in 7 (18%) and 15 (39%), respectively, of 38 patients with sarcoidosis, and in 2 (3%) and 2 (3%), respectively, of 63 patients with other lung diseas‐ es. The results of the study suggested that this antigen from *P. acnes* is responsible for the formation

Response of PBMC to PPD (SI)

(n = 32)

**Figure 16.** Response of peripheral blood mononuclear cells (PBMC) to recombinant trigger factor protein (RP35) from *P. acnes* and purified protein derivative (PPD) from *M. tuberculosis*. The horizontal bars show, from bottom to top, the 25th percentile, median, and 75th percentile, respectively. The dotted lines show the threshold, set at the mean + 3SD

0

**RA**: rheumatoid arthritis

**Sar TB RA Cont**

(n =32)

**Cont**: healthy volunteer

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5

10

15

20

arising from the bacterium gave rise to a Th1 immune response in the subject.

or maintenance of granulomas in some patients with sarcoidosis.

**Sar TB RA Cont**

**TB**: tubercusosis (n = 21)

**6.1. Hypersensitivity to** *P. acnes* **antigens**

Response of PBMC to RP35 (SI)

0

of the 32 control samples.

**Sar**: sarcoidosis (n = 50)

1

2

3

4

5

6

**Figure 14.** A lung sample with many epithelioid cell granulomas with central eosinophilic necrosis. This case required differential diagnosis from tuberculosis although the specimen contained no acid-fast bacilli and the clinical data of the patient suggested sarcoidosis.

**Figure 15.** Immunohistochemistry with the PAB antibody for the specimen shown in Figure 14 revealed positive reac‐ tion products (green arrows) within granulomas accompanied by central eosinophilic necrosis.

consistent with a helper T-cell type 1 (Th1) immune response triggered by undefined antigen(s) [34]. If a propionibacterium caused a particular case of sarcoidosis, it is likely that an antigen arising from the bacterium gave rise to a Th1 immune response in the subject.

#### **6.1. Hypersensitivity to** *P. acnes* **antigens**

**Figure 14.** A lung sample with many epithelioid cell granulomas with central eosinophilic necrosis. This case required differential diagnosis from tuberculosis although the specimen contained no acid-fast bacilli and the clinical data of

**Figure 15.** Immunohistochemistry with the PAB antibody for the specimen shown in Figure 14 revealed positive reac‐

tion products (green arrows) within granulomas accompanied by central eosinophilic necrosis.

the patient suggested sarcoidosis.

16 Sarcoidosis

Ebe and colleagues searched for propionibacterial antigens that evoked cellular immune responses only in patients with sarcoidosis [11]. For this purpose, a λgt11 genomic DNA library of *P. acnes* was screened with sera from patients with sarcoidosis, because high levels of serum antibodies against the antigen usually accompany such an immune response. Of 180,000 plaques screened, 2 clones coded for an identical recombinant protein, RP35, recognized by sera. RP35, a recombinant protein of 256 amino acid residues with a calculated molecular mass of 28,133 Da, is a fragment (the C-terminal region) of the *P. acnes* trigger factor, which has 529 amino acid residues and a calculated molecular mass of 57,614 Da. The C-terminal sequence (Asp-463 to Lys-529) seems to be unique to *P. acnes*, with no similarity to sequences of other bacterial proteins deposited in the Swiss-Prot database. Conformational analysis of the Ser-491 to Lys-529 region at the C terminus revealed it to be highly antigenic. RP35 caused sarcoidosis-specific prolifera‐ tion of peripheral blood mononuclear cells (PBMCs) from 9 (18%) of 50 patients with sarcoido‐ sis (Figure 16). The same study established that serum levels of IgG and IgA antibodies to RP35 are high in patients with sarcoidosis and other lung diseases. In bronchoalveolar lavage (BAL) fluid, IgG and IgA antibody levels were high in 7 (18%) and 15 (39%), respectively, of 38 patients with sarcoidosis, and in 2 (3%) and 2 (3%), respectively, of 63 patients with other lung diseas‐ es. The results of the study suggested that this antigen from *P. acnes* is responsible for the formation or maintenance of granulomas in some patients with sarcoidosis.

**Figure 16.** Response of peripheral blood mononuclear cells (PBMC) to recombinant trigger factor protein (RP35) from *P. acnes* and purified protein derivative (PPD) from *M. tuberculosis*. The horizontal bars show, from bottom to top, the 25th percentile, median, and 75th percentile, respectively. The dotted lines show the threshold, set at the mean + 3SD of the 32 control samples.

Recently, Furusawa *et al* [35] reported that interleukin-2 secretion from PBMCs after stimula‐ tion with viable *P. acnes* is higher in patients with sarcoidosis than in control subjects. Inter‐ leukin-2 and interleukin-12 mRNA expression of PBMCs after stimulation with *P. acnes* is also higher in patients with sarcoidosis than in control subjects. In contrast, interleukin-17 mRNA expression of PBMCs is lower in patients with sarcoidosis than in control subjects. The responses of the two groups to stimulation with *M. tuberculosis* antigens such as Bacille de Calmette et Guérin (BCG) or ESAT-6 recombinant protein were not significantly different. Sarcoidosis may arise from an imbalance of Th1/Th17 immune responses to viable *P. acnes*.

Additional evidence of the hypersensitivity of sarcoid patients to *P. acnes* was obtained in studies of BAL cells. When stimulated with a crude extract of *P. acnes* with pyridine, BAL cells from patients with sarcoidosis proliferated more than BAL cells from healthy subjects or from patients with lung cancer [36]. Interleukin-2 production and interleukin-2 receptor expression of BAL cells stimulated by the *P. acnes* antigen was greater in sarcoidosis patients than in healthy subjects or patients with other lung diseases [37]. *P. acnes* DNA was detected in BAL cells from 21 (70%) of 30 sarcoid patients and 7 (23%) of 30 control patients with other lung diseases [38]. In situ signals of *P. acnes* DNA were detected in the cytoplasm of a few alveolar macrophages among the BAL cells from sarcoid patients, but from no other kinds of BAL cells, including alveolar lymphocytes and neutrophils. Gallium-67 uptake by lung parenchyma was found in about half of the 30 sarcoid patients with *P. acnes* DNA, but in none of the other sarcoid patients [38].

#### **6.2. NOD1 gene polymorphism**

Mutations in the related NOD2 gene predispose patients to granulomatous diseases, including Crohn's disease [39], Blau syndrome [40], and early-onset sarcoidosis [41]. Although Blau syndrome and early-onset sarcoidosis are reported to share identical NOD2 mutations, no association has been reported between NOD2 and sarcoidosis [42]. NOD1 shares many structural and functional similarities with NOD2. Tanabe *et al* found that intracellular *P. acnes* activates NF-κB in both an NOD1- and NOD2-dependent manner [43]. A systematic search for NOD1 gene polymorphisms in Japanese sarcoidosis patients identified two alleles, 796G-haplotype (156C, 483C, 796G, 1722G) and 796A-haplotype (156G, 483T, 796A, 1722A). Allelic discrimination of 73 sarcoidosis patients and 215 healthy individuals showed that the frequency of the 796A-type allele is significantly higher in sarcoidosis patients and the odds ratios (ORs) are significantly elevated in NOD1-796G/A and 796A/A genotypes (OR [95% CI] = 2.250 [1.084, 4.670] and 3.243 [1.402, 7.502], respectively) as compared to the G/G genotype, showing an increasing trend across the 3 genotypes (*P* = 0.006 for trend). Functional studies indicated that the NOD1 796A-allele is associated with reduced expression leading to dimin‐ ished NF-κB activation in response to intracellular *P. acnes* (Figure 17).

during *Helicobacter pylori* infection [46]. It is possible that impaired expression of beta-defen‐ sin-2 through 796-A NOD1 due to a reduced ability to induce NF-κB enables *P. acnes* to survive

**Figure 17.** Functional studies (lower panel) revealed that intracellular *P. acnes* activates NF-κB in a NOD1-dependent manner and the NOD1 796A-allele predominant in sarcoidosis patients causes diminished NF-κB activation in re‐ sponse to intracellular *P. acnes*. Western blot analysis (upper panel) shows reduced expression of the NOD1 796A-al‐

control Wild type Sarcoidosis

0 20 40 0 20 40 (ng)

Wild type Sarcoidosis

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Western blot of Nod1 expression

In experimental animals, granulomatous lesions can be induced by *P. acnes*. A single intrave‐ nous injection of *P. acnes* into mice leads to the development of many granulomas in the liver [47-49], but not in the lungs. Pulmonary granulomas can be induced, however, by an intrave‐ nous injection of *P. acnes* into sensitized rats [50] and rabbits [51]. In these two studies of

and persist intracellularly, leading to the pathogenesis of sarcoidosis.

**7. Experimental models**

**0**

NF-

lele.

kB activity index

**20**

**40**

**(- )** Ligand Invasive

Non- invasive

*P. acnes* has been studied for its role in immunomodulation with the conclusion that Toll-like receptor 2 (TLR2), TLR4, and TLR9 mediate the effects of *P. acnes* infection [44, 45]. These studies, however, only investigated non-invasive *P. acnes*. TLRs are likely to serve as first-line receptors for *P. acnes*, but NOD proteins might play a major role in a subsequent phase of intracellular infection. NOD1 was recently reported to be a critical regulator of beta-defensin-2

**Figure 17.** Functional studies (lower panel) revealed that intracellular *P. acnes* activates NF-κB in a NOD1-dependent manner and the NOD1 796A-allele predominant in sarcoidosis patients causes diminished NF-κB activation in re‐ sponse to intracellular *P. acnes*. Western blot analysis (upper panel) shows reduced expression of the NOD1 796A-al‐ lele.

during *Helicobacter pylori* infection [46]. It is possible that impaired expression of beta-defen‐ sin-2 through 796-A NOD1 due to a reduced ability to induce NF-κB enables *P. acnes* to survive and persist intracellularly, leading to the pathogenesis of sarcoidosis.

### **7. Experimental models**

Recently, Furusawa *et al* [35] reported that interleukin-2 secretion from PBMCs after stimula‐ tion with viable *P. acnes* is higher in patients with sarcoidosis than in control subjects. Inter‐ leukin-2 and interleukin-12 mRNA expression of PBMCs after stimulation with *P. acnes* is also higher in patients with sarcoidosis than in control subjects. In contrast, interleukin-17 mRNA expression of PBMCs is lower in patients with sarcoidosis than in control subjects. The responses of the two groups to stimulation with *M. tuberculosis* antigens such as Bacille de Calmette et Guérin (BCG) or ESAT-6 recombinant protein were not significantly different. Sarcoidosis may arise from an imbalance of Th1/Th17 immune responses to viable *P. acnes*.

Additional evidence of the hypersensitivity of sarcoid patients to *P. acnes* was obtained in studies of BAL cells. When stimulated with a crude extract of *P. acnes* with pyridine, BAL cells from patients with sarcoidosis proliferated more than BAL cells from healthy subjects or from patients with lung cancer [36]. Interleukin-2 production and interleukin-2 receptor expression of BAL cells stimulated by the *P. acnes* antigen was greater in sarcoidosis patients than in healthy subjects or patients with other lung diseases [37]. *P. acnes* DNA was detected in BAL cells from 21 (70%) of 30 sarcoid patients and 7 (23%) of 30 control patients with other lung diseases [38]. In situ signals of *P. acnes* DNA were detected in the cytoplasm of a few alveolar macrophages among the BAL cells from sarcoid patients, but from no other kinds of BAL cells, including alveolar lymphocytes and neutrophils. Gallium-67 uptake by lung parenchyma was found in about half of the 30 sarcoid patients with *P. acnes* DNA, but in none of the other sarcoid

Mutations in the related NOD2 gene predispose patients to granulomatous diseases, including Crohn's disease [39], Blau syndrome [40], and early-onset sarcoidosis [41]. Although Blau syndrome and early-onset sarcoidosis are reported to share identical NOD2 mutations, no association has been reported between NOD2 and sarcoidosis [42]. NOD1 shares many structural and functional similarities with NOD2. Tanabe *et al* found that intracellular *P. acnes* activates NF-κB in both an NOD1- and NOD2-dependent manner [43]. A systematic search for NOD1 gene polymorphisms in Japanese sarcoidosis patients identified two alleles, 796G-haplotype (156C, 483C, 796G, 1722G) and 796A-haplotype (156G, 483T, 796A, 1722A). Allelic discrimination of 73 sarcoidosis patients and 215 healthy individuals showed that the frequency of the 796A-type allele is significantly higher in sarcoidosis patients and the odds ratios (ORs) are significantly elevated in NOD1-796G/A and 796A/A genotypes (OR [95% CI] = 2.250 [1.084, 4.670] and 3.243 [1.402, 7.502], respectively) as compared to the G/G genotype, showing an increasing trend across the 3 genotypes (*P* = 0.006 for trend). Functional studies indicated that the NOD1 796A-allele is associated with reduced expression leading to dimin‐

*P. acnes* has been studied for its role in immunomodulation with the conclusion that Toll-like receptor 2 (TLR2), TLR4, and TLR9 mediate the effects of *P. acnes* infection [44, 45]. These studies, however, only investigated non-invasive *P. acnes*. TLRs are likely to serve as first-line receptors for *P. acnes*, but NOD proteins might play a major role in a subsequent phase of intracellular infection. NOD1 was recently reported to be a critical regulator of beta-defensin-2

ished NF-κB activation in response to intracellular *P. acnes* (Figure 17).

patients [38].

18 Sarcoidosis

**6.2. NOD1 gene polymorphism**

In experimental animals, granulomatous lesions can be induced by *P. acnes*. A single intrave‐ nous injection of *P. acnes* into mice leads to the development of many granulomas in the liver [47-49], but not in the lungs. Pulmonary granulomas can be induced, however, by an intrave‐ nous injection of *P. acnes* into sensitized rats [50] and rabbits [51]. In these two studies of experimental pulmonary granulomas, heat-killed *P. acnes* was used as a sensitizer, and a challenge by a single intravenous injection of the bacterium was essential for granulomas to form in the lungs.

protein, thyroglobulin, and testicular homogenate, respectively) emulsified in CFA, which is essential for the experiment. Autoimmune inflammatory lesions are induced in this way only in the organs from which the self-antigens used for the sensitization originated. In the animal model of sarcoidosis, sensitization of mice with *P. acnes* trigger factor protein or heat-killed *P. acnes* in CFA induces granulomatous inflammation confined to the lungs. This finding suggests

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Similar to the results obtained by bacterial culture of human samples from lung and lymph nodes, *P. acnes* was cultured from the lungs, liver, and lymph nodes from some of the untreated normal mice, and culture was most often successful with the lungs. There was an unexpected concordance in the rate (33%) of culture from normal lungs and the frequency of detection of pulmonary granulomas in mice sensitized with the trigger factor protein. The concordance suggests that mice without granulomas may have been free from *P. acnes* in the normal

Using the same experimental protocol with rabbits bred in a conventional environment, rabbits sensitized with the *P. acnes* trigger factor antigen developed more severe and diffuse pulmo‐ nary granulomatosis than did sensitized mice. In fact, the severe granulomatous inflammation that developed could even be identified macroscopically (Figure 19, 20). Th1 immune response to the *P. acnes* trigger factor protein might have caused pulmonary granulomas at the sites of *P. acnes* infection. Indeed, the administration of antibiotics (azithromycin in mice and mino‐ cycline in rabbits) before and during the experiments prevented granuloma formation in these

**Figure 19.** Cut sections of the lungs from a rabbit with experimental pulmonary granulomatosis induced by sensitiza‐ tion with *P. acnes* trigger factor protein and adjuvant. Whitish lesions are distributed throughout and are especially

that such antigens from *P. acnes* exist in the lungs of mice even before the experiment.

indigenous flora of their lungs before and during the experiment.

experimental models (Figure 21).

prominent in the subpleural and interlobular areas.

#### **7.1. Pulmonary granulomatosis caused by sensitization with** *P. acnes* **antigens**

*P. acnes* trigger factor protein (RP35) or heat-killed *P. acnes* causes pulmonary granulomas in some (25%-57%) mice sensitized with the protein and complete Freund's adjuvant (CFA) [52]. An intravenous injection of *P. acnes* as a challenge was not essential for granulomas to form in the lungs. Granulomas were scattered throughout the lungs, especially in subpleural areas (Figure 18). The granulomas were composed of a core of epithelioid cells intermingled with a few and surrounded by many mononuclear cells. The detection frequency of pulmonary granulomas did not differ significantly between mice sensitized with the RP35 or heat-killed *P. acnes*.

**Figure 18.** A non-caseating epithelioid cell granuloma observed in a mouse with experimental pulmonary granuloma‐ tosis induced by sensitization with *P. acnes* trigger factor protein and adjuvant.

This experimental protocol may provide a satisfactory model of sarcoidosis. First, hypersen‐ sitivity to *P. acnes* trigger factor, such as has been experimentally induced, has been reported in some patients with sarcoidosis. Second, situations resembling intravenous challenge with *P. acnes* are rare in humans, and sarcoidosis can start in asymptomatic persons without evidence of septicemia.

Experimental models of allergic diseases, such as encephalomyelitis [53], thyroiditis [54], and orchitis [55] have been produced by immunizing animals with self-antigens (myelin basic protein, thyroglobulin, and testicular homogenate, respectively) emulsified in CFA, which is essential for the experiment. Autoimmune inflammatory lesions are induced in this way only in the organs from which the self-antigens used for the sensitization originated. In the animal model of sarcoidosis, sensitization of mice with *P. acnes* trigger factor protein or heat-killed *P. acnes* in CFA induces granulomatous inflammation confined to the lungs. This finding suggests that such antigens from *P. acnes* exist in the lungs of mice even before the experiment.

experimental pulmonary granulomas, heat-killed *P. acnes* was used as a sensitizer, and a challenge by a single intravenous injection of the bacterium was essential for granulomas to

*P. acnes* trigger factor protein (RP35) or heat-killed *P. acnes* causes pulmonary granulomas in some (25%-57%) mice sensitized with the protein and complete Freund's adjuvant (CFA) [52]. An intravenous injection of *P. acnes* as a challenge was not essential for granulomas to form in the lungs. Granulomas were scattered throughout the lungs, especially in subpleural areas (Figure 18). The granulomas were composed of a core of epithelioid cells intermingled with a few and surrounded by many mononuclear cells. The detection frequency of pulmonary granulomas did not differ significantly between mice sensitized with the RP35 or heat-killed

**Figure 18.** A non-caseating epithelioid cell granuloma observed in a mouse with experimental pulmonary granuloma‐

This experimental protocol may provide a satisfactory model of sarcoidosis. First, hypersen‐ sitivity to *P. acnes* trigger factor, such as has been experimentally induced, has been reported in some patients with sarcoidosis. Second, situations resembling intravenous challenge with *P. acnes* are rare in humans, and sarcoidosis can start in asymptomatic persons without

Experimental models of allergic diseases, such as encephalomyelitis [53], thyroiditis [54], and orchitis [55] have been produced by immunizing animals with self-antigens (myelin basic

tosis induced by sensitization with *P. acnes* trigger factor protein and adjuvant.

evidence of septicemia.

**7.1. Pulmonary granulomatosis caused by sensitization with** *P. acnes* **antigens**

form in the lungs.

20 Sarcoidosis

*P. acnes*.

Similar to the results obtained by bacterial culture of human samples from lung and lymph nodes, *P. acnes* was cultured from the lungs, liver, and lymph nodes from some of the untreated normal mice, and culture was most often successful with the lungs. There was an unexpected concordance in the rate (33%) of culture from normal lungs and the frequency of detection of pulmonary granulomas in mice sensitized with the trigger factor protein. The concordance suggests that mice without granulomas may have been free from *P. acnes* in the normal indigenous flora of their lungs before and during the experiment.

Using the same experimental protocol with rabbits bred in a conventional environment, rabbits sensitized with the *P. acnes* trigger factor antigen developed more severe and diffuse pulmo‐ nary granulomatosis than did sensitized mice. In fact, the severe granulomatous inflammation that developed could even be identified macroscopically (Figure 19, 20). Th1 immune response to the *P. acnes* trigger factor protein might have caused pulmonary granulomas at the sites of *P. acnes* infection. Indeed, the administration of antibiotics (azithromycin in mice and mino‐ cycline in rabbits) before and during the experiments prevented granuloma formation in these experimental models (Figure 21).

**Figure 19.** Cut sections of the lungs from a rabbit with experimental pulmonary granulomatosis induced by sensitiza‐ tion with *P. acnes* trigger factor protein and adjuvant. Whitish lesions are distributed throughout and are especially prominent in the subpleural and interlobular areas.

previous study [52]. In the study, *P. acnes* was identified in normal murine alveolar cells by immunostaining with a *P. acnes*-specific monoclonal antibody (PAB antibody). *P. acnes* was taken up by lung cells, and the *P. acnes*-bearing cells expressed F4/80 rather than CD11c or DEC205, consistent with the ability of macrophages to phagocytose and deliver antigens to dendritic cells in the lung. As far away as the end of the airway, airborne organisms are impacted and eliminated by mechanical defenses, including mucocilliary clearance and coughing. Nevertheless, a small number of *P. acnes* might escape this system and reside on the alveolar surface. *P. acnes* genomes were detected in normal pulmonary lymph nodes as well as the lungs, and lymphocytes from lymph nodes showed *P. acnes*-specific proliferation, suggesting that these cells had already been exposed to *P. acnes* by lung-derived antigenpresenting-cells and had established a memory response. Additionally, these results indicate that *P. acnes* were continuously transported to pulmonary regional lymph nodes in the steady state. Because of this constant delivery of antigens to the pulmonary lymph nodes for a long period, the small number of indigenous *P. acnes* in the normal lung would be enough to produce a specific immune response, but not for the formation of a steady-state granuloma. Although mycobacterial, atypical mycobacterial, and other propionibacterial antigens are potential candidate endogenous microorganisms that trigger pulmonary granuloma forma‐ tion, genomic analyses revealed an absence of these organisms in the lungs of specific-

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The adoptive transfer of *P. acnes*-sensitized lymph node CD4+ T cells into naïve mice resulted in granulomatous changes in the lung, indicating that extrapulmonary lymph node CD4+ T cells primed with *P. acnes* can interact with pulmonary resident cells via the circulation and induce granuloma formation in the normal lung. It was therefore hypothe‐ sized that a continuous supply of *P. acnes*-sensitized T cells should lead to chronic pulmo‐ nary granuloma formation, and consequently we performed continuous remote sensitization of normal mice with *P. acnes*. These mice exhibited distinct pulmonary granulomas, distributed in lymph-rich spaces such as the subpleural, peribronchial, and perivascular areas, and had typical cellular components of granuloma and preferential Th1 cytokine expression. These features are similar to those of pulmonary sarcoidosis. In addition, the ratio of CD4 to CD8 BAL lymphocytes was elevated in the group immunized twice, and serum calcium levels were also increased. Thus, the characteristics of this *P. acnes*-immuni‐ zation model, without any direct exposure of antigen to the lung, showed several similari‐

That study also examined whether changes in the number of pre-existing *P. acnes* cells in the lung affected pulmonary granuloma formation. As expected, preloading of *P. acnes* exacer‐ bated pulmonary disorders, whereas reduction of the *P. acnes* population by antimicrobial treatment reduced the pulmonary lesions. These findings suggest a pivotal role of normally localized *P. acnes* in the formation of pulmonary granuloma by extrapulmonary *P. acnes* sensitization, as well as the potential clinical usefulness of antimicrobial eradication targeting lung-indigenous *P. acnes* for the treatment of pulmonary granulomatosis induced by similar

pathogen-free C57BL/6 mice.

ties to those of sarcoid patients.

pathogenesis.

**Figure 20.** Histologic features of experimental pulmonary granulomatosis of the rabbit shown in Figure 19. Multiple non-caseating epithelioid cell granulomas are accompanied by surrounding lymphoid cell infiltration with alveolitis.

**Figure 21.** Prevention of pulmonary granulomatosis in mice and rabbits sensitized with *P. acnes* trigger factor (TIG) antigen by administration of antibiotics before and during the experiments.

#### **7.2. Mechanism of granuloma formation in the experimental models**

Nishiwaki *et al* further examined the mechanism of pulmonary granulomatosis caused by sensitization of heat-killed *P. acnes* [56], using a similar experimental protocol as used in a previous study [52]. In the study, *P. acnes* was identified in normal murine alveolar cells by immunostaining with a *P. acnes*-specific monoclonal antibody (PAB antibody). *P. acnes* was taken up by lung cells, and the *P. acnes*-bearing cells expressed F4/80 rather than CD11c or DEC205, consistent with the ability of macrophages to phagocytose and deliver antigens to dendritic cells in the lung. As far away as the end of the airway, airborne organisms are impacted and eliminated by mechanical defenses, including mucocilliary clearance and coughing. Nevertheless, a small number of *P. acnes* might escape this system and reside on the alveolar surface. *P. acnes* genomes were detected in normal pulmonary lymph nodes as well as the lungs, and lymphocytes from lymph nodes showed *P. acnes*-specific proliferation, suggesting that these cells had already been exposed to *P. acnes* by lung-derived antigenpresenting-cells and had established a memory response. Additionally, these results indicate that *P. acnes* were continuously transported to pulmonary regional lymph nodes in the steady state. Because of this constant delivery of antigens to the pulmonary lymph nodes for a long period, the small number of indigenous *P. acnes* in the normal lung would be enough to produce a specific immune response, but not for the formation of a steady-state granuloma. Although mycobacterial, atypical mycobacterial, and other propionibacterial antigens are potential candidate endogenous microorganisms that trigger pulmonary granuloma forma‐ tion, genomic analyses revealed an absence of these organisms in the lungs of specificpathogen-free C57BL/6 mice.

The adoptive transfer of *P. acnes*-sensitized lymph node CD4+ T cells into naïve mice resulted in granulomatous changes in the lung, indicating that extrapulmonary lymph node CD4+ T cells primed with *P. acnes* can interact with pulmonary resident cells via the circulation and induce granuloma formation in the normal lung. It was therefore hypothe‐ sized that a continuous supply of *P. acnes*-sensitized T cells should lead to chronic pulmo‐ nary granuloma formation, and consequently we performed continuous remote sensitization of normal mice with *P. acnes*. These mice exhibited distinct pulmonary granulomas, distributed in lymph-rich spaces such as the subpleural, peribronchial, and perivascular areas, and had typical cellular components of granuloma and preferential Th1 cytokine expression. These features are similar to those of pulmonary sarcoidosis. In addition, the ratio of CD4 to CD8 BAL lymphocytes was elevated in the group immunized twice, and serum calcium levels were also increased. Thus, the characteristics of this *P. acnes*-immuni‐ zation model, without any direct exposure of antigen to the lung, showed several similari‐ ties to those of sarcoid patients.

**Figure 20.** Histologic features of experimental pulmonary granulomatosis of the rabbit shown in Figure 19. Multiple non-caseating epithelioid cell granulomas are accompanied by surrounding lymphoid cell infiltration with alveolitis.

untreated treated with

minocycline

Mice sensitized with TIG antigen Rabbits sensitized with TIG antigen

300

0

**Figure 21.** Prevention of pulmonary granulomatosis in mice and rabbits sensitized with *P. acnes* trigger factor (TIG)

Nishiwaki *et al* further examined the mechanism of pulmonary granulomatosis caused by sensitization of heat-killed *P. acnes* [56], using a similar experimental protocol as used in a

100

1000

200

200

**0**

0

untreated treated with

antigen by administration of antibiotics before and during the experiments.

<sup>300</sup> 10080

azithtomycin

**7.2. Mechanism of granuloma formation in the experimental models**

**20**

20

**40**

40

**60**

60

Number of granulomas of the lung

22 Sarcoidosis

**80**

**100**

That study also examined whether changes in the number of pre-existing *P. acnes* cells in the lung affected pulmonary granuloma formation. As expected, preloading of *P. acnes* exacer‐ bated pulmonary disorders, whereas reduction of the *P. acnes* population by antimicrobial treatment reduced the pulmonary lesions. These findings suggest a pivotal role of normally localized *P. acnes* in the formation of pulmonary granuloma by extrapulmonary *P. acnes* sensitization, as well as the potential clinical usefulness of antimicrobial eradication targeting lung-indigenous *P. acnes* for the treatment of pulmonary granulomatosis induced by similar pathogenesis.

### **8. Etiology of sarcoidosis**

In the past, once the germ theory of disease was accepted, microbes were considered to be pathogens if they met the stipulations of Koch's postulates. Although there are many microbes, however, most human infections are caused by only a few. Some microbes have been classified as pathogens although they do not cause disease in every host. In addition, some microbes have been classified as nonpathogenic although they cause disease in certain hosts. For these reasons, in a redefinition of the concepts of virulence and pathogenicity of microbes, Casade‐ vall and Pirofski suggested a classification system for pathogens based on their ability to cause damage as a function of the host's immune response [57]. Koch's postulates for exogenous infection cannot be applied to diseases caused by endogenous bacteria. Endogenous infection is a disease caused by indigenous microorganisms. According to the classification system suggested by Casadevall and Pirofski, endogenous infection, which does not cause any lesions under normal immune conditions, can be classified into three major categories (Figure 22). Opportunistic infections, such as pneumocystis carinii pneumonia, are well known to be associated with immunodeficiency in AIDS patients. Combination type infections, such as Candida and Aspergillus, not only cause opportunistic infections, but may also cause hyper‐ sensitivity pneumonitis. The hypersensitivity type of endogenous infection does not cause any tissue damage until the hypersensitive immune response is triggered. *P. acnes* as a cause of sarcoidosis can therefore be classified within the group of endogenous diseases that results from hypersensitivity.

Occasional detection of intracellular *P. acnes* in non-granulomatous areas of the lungs and lymph nodes from non-sarcoid patients suggests that latent infection and endogenous reactivation of this indigenous bacterium occurs in these organs, even in patients without sarcoidosis. Sarcoidosis involves many organs, and the lungs and mediastinal lymph nodes are involved at the highest frequency [58]. Commensalism of *P. acnes* in these organs may

*P. acnes*, indigenous low-virulence bacterium, can cause latent infection in the lungs and lymph nodes and persist in a cell-wall-deficient form. This dormant form of *P. acnes* can be activated endogenously under certain environmental conditions and then proliferate in cells at the site of the latent infection. In patients hypersensitive to this endogenous bacterium, granulomatous inflammation is triggered by intracellular proliferation of the bacterium. Some proliferating bacteria may escape from isolation by the granuloma and spread to other organs via the lymph and blood streams. The spread of infective *P. acnes* might cause a new latent infection in systemic organs, such as eyes, skin, and heart. Latent infection established in certain systemic organs will be reactivated simultaneously by the next triggering event, resulting in the onset

> Inapparent infection via the respiratory tract

**Figure 23.** Hypothesized mechanism of systemic sarcoid granuloma formation caused by *P. acnes*.

Intracellular proliferation of *P. acnes* triggered by endogenous activation of latent infection might lead to the spread of the infectious *P. acnes*, giving rise to a new latent infection even within the same organ. As long as such latent infection is inadequately eradicated by the host defense mechanism of granuloma formation, the process will be repeated anytime reactivation occurs under the requisite environmental conditions. Relapsing sarcoidosis causes repetitive acute inflammation and post-inflammatory scars in the affected organs, which results in the progres‐ sion of sarcoidosis through tissue damage and functional disorder in the affected organ.

Eyes, Skin, Heart, Liver, Spleen, Muscle, CNS, etc.

Primary infection

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via the lymphatic and blood streams

Spread of infective *P. acnes*

explain why they are frequently involved in sarcoidosis.

**8.2. Mechanism of granuloma formation in sarcoidosis**

of systemic sarcoidosis (Figure 23).

Intracellular proliferation

Latent infection

Lungs & hilar lymph nodes

Granuloma formation

Hypersensitivity

Endogenous activation

: Normal range of immune response in healthy subjects

**Figure 22.** Three major categories of endogenous infection in the classification system of diseases caused by indige‐ nous microorganisms.

#### **8.1. Commensalism of** *P. acnes* **in the lungs and lymph nodes**

*P. acnes* is the most common commensal bacterium in the lungs and lymph nodes from subjects without sarcoidosis [17]. Some *P. acnes* is found in 20% of non-sarcoid lymph nodes by bacterial culture [17], 15% of non-sarcoid lymph nodes by PCR [26], and 18% of non-sarcoid lung samples, and 22% of non-sarcoid lymph node samples by immunohistochemistry [28]. Occasional detection of intracellular *P. acnes* in non-granulomatous areas of the lungs and lymph nodes from non-sarcoid patients suggests that latent infection and endogenous reactivation of this indigenous bacterium occurs in these organs, even in patients without sarcoidosis. Sarcoidosis involves many organs, and the lungs and mediastinal lymph nodes are involved at the highest frequency [58]. Commensalism of *P. acnes* in these organs may explain why they are frequently involved in sarcoidosis.

#### **8.2. Mechanism of granuloma formation in sarcoidosis**

**8. Etiology of sarcoidosis**

24 Sarcoidosis

from hypersensitivity.

nous microorganisms.

**Opportunistic infection** *(Pneumocystis carinii)*

Normal range

In the past, once the germ theory of disease was accepted, microbes were considered to be pathogens if they met the stipulations of Koch's postulates. Although there are many microbes, however, most human infections are caused by only a few. Some microbes have been classified as pathogens although they do not cause disease in every host. In addition, some microbes have been classified as nonpathogenic although they cause disease in certain hosts. For these reasons, in a redefinition of the concepts of virulence and pathogenicity of microbes, Casade‐ vall and Pirofski suggested a classification system for pathogens based on their ability to cause damage as a function of the host's immune response [57]. Koch's postulates for exogenous infection cannot be applied to diseases caused by endogenous bacteria. Endogenous infection is a disease caused by indigenous microorganisms. According to the classification system suggested by Casadevall and Pirofski, endogenous infection, which does not cause any lesions under normal immune conditions, can be classified into three major categories (Figure 22). Opportunistic infections, such as pneumocystis carinii pneumonia, are well known to be associated with immunodeficiency in AIDS patients. Combination type infections, such as Candida and Aspergillus, not only cause opportunistic infections, but may also cause hyper‐ sensitivity pneumonitis. The hypersensitivity type of endogenous infection does not cause any tissue damage until the hypersensitive immune response is triggered. *P. acnes* as a cause of sarcoidosis can therefore be classified within the group of endogenous diseases that results

: Normal range of immune response in healthy subjects

**Hypersensitivity** *(P. acnes)*

Immune Response Immune Response Immune Response

**Figure 22.** Three major categories of endogenous infection in the classification system of diseases caused by indige‐

*P. acnes* is the most common commensal bacterium in the lungs and lymph nodes from subjects without sarcoidosis [17]. Some *P. acnes* is found in 20% of non-sarcoid lymph nodes by bacterial culture [17], 15% of non-sarcoid lymph nodes by PCR [26], and 18% of non-sarcoid lung samples, and 22% of non-sarcoid lymph node samples by immunohistochemistry [28].

**8.1. Commensalism of** *P. acnes* **in the lungs and lymph nodes**

**Combination type** *(Candida & Aspergillus)* *P. acnes*, indigenous low-virulence bacterium, can cause latent infection in the lungs and lymph nodes and persist in a cell-wall-deficient form. This dormant form of *P. acnes* can be activated endogenously under certain environmental conditions and then proliferate in cells at the site of the latent infection. In patients hypersensitive to this endogenous bacterium, granulomatous inflammation is triggered by intracellular proliferation of the bacterium. Some proliferating bacteria may escape from isolation by the granuloma and spread to other organs via the lymph and blood streams. The spread of infective *P. acnes* might cause a new latent infection in systemic organs, such as eyes, skin, and heart. Latent infection established in certain systemic organs will be reactivated simultaneously by the next triggering event, resulting in the onset of systemic sarcoidosis (Figure 23).

**Figure 23.** Hypothesized mechanism of systemic sarcoid granuloma formation caused by *P. acnes*.

Intracellular proliferation of *P. acnes* triggered by endogenous activation of latent infection might lead to the spread of the infectious *P. acnes*, giving rise to a new latent infection even within the same organ. As long as such latent infection is inadequately eradicated by the host defense mechanism of granuloma formation, the process will be repeated anytime reactivation occurs under the requisite environmental conditions. Relapsing sarcoidosis causes repetitive acute inflammation and post-inflammatory scars in the affected organs, which results in the progres‐ sion of sarcoidosis through tissue damage and functional disorder in the affected organ.

Sarcoidosis is most likely the result of a complex interaction between infection, immunity, and allergic reaction (Figure 24). There are three conditions essential to the development of sarcoidosis caused by *P. acnes*: 1) latent infection with cell-wall-deficient *P. acnes*, 2) endoge‐ nous activation of dormant *P. acnes* triggered by certain environmental factors, and 3) a hypersensitive Th1 immune response towards the intracellular proliferation of *P. acnes*. The formation of sarcoid granulomas might be induced by a Th1 immune response to one or more antigens of *P. acnes* proliferating in the affected organ or tissue in an individual with a hereditary or acquired abnormality of the immune system.

latent tuberculosis infection [59, 60]. Tuberculosis and sarcoidosis are side effects of the antitumor necrosis factor-α drugs administered to patients with rheumatoid arthritis [61, 62]. Antitumor necrosis factor-α treatment is thought to reactivate latent tuberculosis infection [63]. In the same manner, latent *P. acnes* might be reactivated by anti- tumor necrosis factor-α treat‐

Immunosuppressive, mainly corticosteroidal, therapy has been used for sarcoidosis for more than 50 years, but the long-term effects of steroidal treatment in chronic sarcoidosis are still disputed [3], Further, the high relapse rate after treatment and the side effects of long-term use are often a clinical challenge [64]. Steroids suppress the allergic reaction, thereby providing therapeutic effects. Interference of the inflammatory process by these immunosuppressive drugs, however, may impede the formation of granulomas, which function to curtail the

Antibiotics not only kill the bacteria proliferating in cells, but also prevent the endogenous activation of latent bacteria. Long-term administration of antibiotics may therefore be effective for patients with progressive sarcoidosis by preventing inflammatory relapses caused by reactivation of the latent bacteria (Figure 25). If latent bacterial infection persisting in organs can be eradicated by treatment with antibiotics, complete remission of sarcoidosis may be achieved. Complete eradication of latent bacteria might be difficult to achieve through the

> Prevention of endogenous activation

Antibiotics

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Corticosteroids

Eradication of latent bacteria

Prevention of intracellular proliferation

Suppression of allergic reaction

conventional use of antibiotics, however, as in the case of pulmonary tuberculosis.

ment, resulting in sarcoidosis in certain susceptible subjects among these patients.

**9. Treatment of sarcoidosis**

spread of *P. acnes* infection.

**9.1. Strategy for treating sarcoidosis caused by P. acnes**

Endogenous activation (Intracellular proliferation)

Environmental factors

Latent infection (cell-wall-deficient form)

Granuloma formation

**Figure 25.** Strategy for treating sarcoidosis caused by *P. acnes*.

Th1 hypersensitivity

**Figure 24.** Sarcoidosis caused by *P. acnes* with a complex interaction of latent infection, endogenous activation, and host hypersensitivity.

#### **8.3. Pathogenesis shared by sarcoidosis and tuberculosis**

Tuberculosis shares many common features with sarcoidosis, not only their histopathologic features, but also aspects of their pathogenesis. Many tuberculosis cases arise from the endogenous activation of latent tuberculosis infection. Primary *M. tuberculosis* infection usually occurs in childhood and produces lesions termed the "primary complex", which is a combination of lesions in the lung and lung hilar lymph nodes. Around 90% of subjects with primary infection by *M. tuberculosis* exhibit a so-called "unapparent infection", i.e., they are asymptomatic. Latent infection by this pathogen is characterized by healed lesions comprising consolidated scar tissue or necrotic lesions that often becomes calcified. The persistent mycobacteria in this dormant phase are thought to be cell-wall-deficient. Active tuberculosis occurs when the latent mycobacterial infection is endogenously activated under certain environmental conditions, especially in older people. The risk for activation is also significantly increased by immunosuppressive triggers, such as HIV infection and diabetes. Recent immunologic data provide evidence of latent tuberculosis in about one-third of the global population, which corresponds to more than 2 billion individuals [59].

Endogenous reactivation of latent bacteria is well known to occur in tuberculosis, which shares many common features with sarcoidosis, not only the histopathologic features, but also the pathogenic features. Many cases of adult tuberculosis are caused by endogenous activation of latent tuberculosis infection [59, 60]. Tuberculosis and sarcoidosis are side effects of the antitumor necrosis factor-α drugs administered to patients with rheumatoid arthritis [61, 62]. Antitumor necrosis factor-α treatment is thought to reactivate latent tuberculosis infection [63]. In the same manner, latent *P. acnes* might be reactivated by anti- tumor necrosis factor-α treat‐ ment, resulting in sarcoidosis in certain susceptible subjects among these patients.

### **9. Treatment of sarcoidosis**

Sarcoidosis is most likely the result of a complex interaction between infection, immunity, and allergic reaction (Figure 24). There are three conditions essential to the development of sarcoidosis caused by *P. acnes*: 1) latent infection with cell-wall-deficient *P. acnes*, 2) endoge‐ nous activation of dormant *P. acnes* triggered by certain environmental factors, and 3) a hypersensitive Th1 immune response towards the intracellular proliferation of *P. acnes*. The formation of sarcoid granulomas might be induced by a Th1 immune response to one or more antigens of *P. acnes* proliferating in the affected organ or tissue in an individual with a

Sarcoidosis

**Figure 24.** Sarcoidosis caused by *P. acnes* with a complex interaction of latent infection, endogenous activation, and

Tuberculosis shares many common features with sarcoidosis, not only their histopathologic features, but also aspects of their pathogenesis. Many tuberculosis cases arise from the endogenous activation of latent tuberculosis infection. Primary *M. tuberculosis* infection usually occurs in childhood and produces lesions termed the "primary complex", which is a combination of lesions in the lung and lung hilar lymph nodes. Around 90% of subjects with primary infection by *M. tuberculosis* exhibit a so-called "unapparent infection", i.e., they are asymptomatic. Latent infection by this pathogen is characterized by healed lesions comprising consolidated scar tissue or necrotic lesions that often becomes calcified. The persistent mycobacteria in this dormant phase are thought to be cell-wall-deficient. Active tuberculosis occurs when the latent mycobacterial infection is endogenously activated under certain environmental conditions, especially in older people. The risk for activation is also significantly increased by immunosuppressive triggers, such as HIV infection and diabetes. Recent immunologic data provide evidence of latent tuberculosis in about one-third of the global

Endogenous reactivation of latent bacteria is well known to occur in tuberculosis, which shares many common features with sarcoidosis, not only the histopathologic features, but also the pathogenic features. Many cases of adult tuberculosis are caused by endogenous activation of

Hypersensitivity caused by host factors

Latent infection of cell-wall-deficient *P. acnes*

hereditary or acquired abnormality of the immune system.

Endogenous activation triggered by environmental factors

**8.3. Pathogenesis shared by sarcoidosis and tuberculosis**

population, which corresponds to more than 2 billion individuals [59].

host hypersensitivity.

26 Sarcoidosis

Immunosuppressive, mainly corticosteroidal, therapy has been used for sarcoidosis for more than 50 years, but the long-term effects of steroidal treatment in chronic sarcoidosis are still disputed [3], Further, the high relapse rate after treatment and the side effects of long-term use are often a clinical challenge [64]. Steroids suppress the allergic reaction, thereby providing therapeutic effects. Interference of the inflammatory process by these immunosuppressive drugs, however, may impede the formation of granulomas, which function to curtail the spread of *P. acnes* infection.

#### **9.1. Strategy for treating sarcoidosis caused by P. acnes**

Antibiotics not only kill the bacteria proliferating in cells, but also prevent the endogenous activation of latent bacteria. Long-term administration of antibiotics may therefore be effective for patients with progressive sarcoidosis by preventing inflammatory relapses caused by reactivation of the latent bacteria (Figure 25). If latent bacterial infection persisting in organs can be eradicated by treatment with antibiotics, complete remission of sarcoidosis may be achieved. Complete eradication of latent bacteria might be difficult to achieve through the conventional use of antibiotics, however, as in the case of pulmonary tuberculosis.

**Figure 25.** Strategy for treating sarcoidosis caused by *P. acnes*.

Another approach for treating sarcoidosis is specific suppression of the hypersensitivity to *P. acnes* antigens responsible for granuloma formation, such as the trigger factor protein. Recent advances in the modulation of epitope-specific immune responses by synthetic peptides might help to improve the treatment of sarcoidosis.

*acnes* proposed in this chapter suggest that these antibiotics have many roles in the process of granuloma formation, including the events triggering granuloma formation caused by

*Propionibacterium acnes* as a Cause of Sarcoidosis

http://dx.doi.org/10.5772/55073

29

The Marshall protocol [72] is an eradication therapy for intracellular bacteria established by Dr. Trevor Marshall. This therapeutic protocol is a combination of minocycline plus azithro‐ mycin or clindamycin, supported by the use of an angiotensin receptor blocker to prevent Herxheimer reactions. According to the results published by the Autoimmunity Research Foundation in 2006, this therapy is effective in 62% of patients with sarcoidosis. Information about the treatment can be found at the study site, marshallprotocol.com and also at autoim‐

Department of Human Pathology, Division of Surgical Pathology, Tokyo Medical and Den‐

[1] Newman LS, Rose CS, Maier LA. Sarcoidosis. N Engl J Med 1997;336:1224-34.

[3] Baughman RP, Lower EE, du Bois RM. Sarcoidosis. Lancet 2003;361:1111-8.

and Other Granulomatous Disorders 1999;16:149-73.

[2] Hunninghake GW, Costabel U, Ando M, et al. ATS/ERS/WASOG statement on sar‐ coidosis. American Thoracic Society/European Respiratory Society/World Associa‐ tion of Sarcoidosis and other Granulomatous Disorders. Sarcoidosis, vasculitis, and diffuse lung diseases : official journal of WASOG / World Association of Sarcoidosis

[4] Newman LS, Rose CS, Bresnitz EA, et al. A case control etiologic study of sarcoido‐ sis: environmental and occupational risk factors. Am J Respir Crit Care Med

[5] McGrath DS, Goh N, Foley PJ, et al. Sarcoidosis: genes and microbes--soil or seed? Sarcoidosis, vasculitis, and diffuse lung diseases : official journal of WASOG / World Association of Sarcoidosis and Other Granulomatous Disorders 2001;18:149-64.

intracellular proliferation of the bacterium.

Address all correspondence to: eishi.path@tmd.ac.jp

**9.3. Marshall protocol**

munityresearch.org.

**Author details**

Yoshinobu Eishi\*

tal University, Japan

2004;170:1324-30.

**References**

#### **9.2. Tetracyclines for treating sarcoidosis**

Minocycline is the first-choice antibiotic for patients with acne vulgaris caused by *P. acnes*. In an observational study, Bachelez and colleagues [65] reported the possible benefits of tetra‐ cyclines, to which *P. acnes* is sensitive, for the treatment of chronic forms of cutaneous sarcoidosis. The authors treated 12 patients with biopsy-proven cutaneous sarcoidosis with 200 mg daily minocycline over a median 12-month period. With a median follow-up of 26 months, the authors noted complete and partial responses to treatment in 8 and 2 patients, respectively. The mean time to reach a maximal response was 3.2 months. Of the 8 patients who had a complete response, minocycline could be withdrawn in 7 patients, 3 of whom experienced recurrent lesions and received further treatment with 200 mg daily doxycycline. Complete remission was maintained for a mean of 15.3 months. Regression of pulmonary infiltrates and mediastinal lymphadenopathy was noted in the 2 patients with concurrent pulmonary involvement.

The results of a nationwide questionnaire survey, performed by a Japanese research group in 2005 (reported in Japanese), indicated that antibiotic therapy was effective in 43% of 87 patients with sarcoidosis treated with many kinds of antibiotics, including minocycline, doxycycline, and clarithromycin. Baba and colleagues [66] used minocycline and clarithromycin for therapy against worsening of multiple endobronchial mass lesions, given the possible roles of *P. acnes* in the pathophysiology of sarcoidosis and the uncertainty of the long-term effects of corticosteroids. The lesions were worsening before the antibiotic therapy was initiated, so the improvements appeared to be due to the therapy. Park and colleagues [67] reported the first case of ocular and ocular adnexal sarcoidosis treated with minocycline. The patient in this report demonstrated a complete and recurrence-free response of lacrimal gland and choroidal lesions as well as parotid granulomas and pulmonary infiltrates after 3 months of minocycline therapy. Miyazaki and colleagues [68] reported the regression of nodular lesions of muscular sarcoidosis during minocycline treatment with decreased levels of angiotensin-converting enzyme, lysozyme, and soluble interleukin-2 receptor. The effectiveness of minocycline for muscular sarcoidosis in this case was confirmed when a prompt response to the minocycline therapy was repeatedly observed.

Based on the studies described in this section, the antimicrobial properties of tetracyclines are effective for treating sarcoidosis. Many researchers, have questioned the antimicrobial role of tetracyclines because tetracyclines also have anti-inflammatory properties, which were demonstrated by in vitro studies and corroborated by clinical trials. Tetracycline suppresses neutrophil migration and chemotaxis [69], and minocycline inhibits T-lymphocyte activation and proliferation [70]. Both minocycline and doxycycline obviate granuloma formation in vitro [71]. Although it remains controversial whether these antibiotics kill microbes or have only an anti-inflammatory effect, the mechanisms of sarcoid granuloma formation caused by *P.* *acnes* proposed in this chapter suggest that these antibiotics have many roles in the process of granuloma formation, including the events triggering granuloma formation caused by intracellular proliferation of the bacterium.

#### **9.3. Marshall protocol**

Another approach for treating sarcoidosis is specific suppression of the hypersensitivity to *P. acnes* antigens responsible for granuloma formation, such as the trigger factor protein. Recent advances in the modulation of epitope-specific immune responses by synthetic peptides might

Minocycline is the first-choice antibiotic for patients with acne vulgaris caused by *P. acnes*. In an observational study, Bachelez and colleagues [65] reported the possible benefits of tetra‐ cyclines, to which *P. acnes* is sensitive, for the treatment of chronic forms of cutaneous sarcoidosis. The authors treated 12 patients with biopsy-proven cutaneous sarcoidosis with 200 mg daily minocycline over a median 12-month period. With a median follow-up of 26 months, the authors noted complete and partial responses to treatment in 8 and 2 patients, respectively. The mean time to reach a maximal response was 3.2 months. Of the 8 patients who had a complete response, minocycline could be withdrawn in 7 patients, 3 of whom experienced recurrent lesions and received further treatment with 200 mg daily doxycycline. Complete remission was maintained for a mean of 15.3 months. Regression of pulmonary infiltrates and mediastinal lymphadenopathy was noted in the 2 patients with concurrent

The results of a nationwide questionnaire survey, performed by a Japanese research group in 2005 (reported in Japanese), indicated that antibiotic therapy was effective in 43% of 87 patients with sarcoidosis treated with many kinds of antibiotics, including minocycline, doxycycline, and clarithromycin. Baba and colleagues [66] used minocycline and clarithromycin for therapy against worsening of multiple endobronchial mass lesions, given the possible roles of *P. acnes* in the pathophysiology of sarcoidosis and the uncertainty of the long-term effects of corticosteroids. The lesions were worsening before the antibiotic therapy was initiated, so the improvements appeared to be due to the therapy. Park and colleagues [67] reported the first case of ocular and ocular adnexal sarcoidosis treated with minocycline. The patient in this report demonstrated a complete and recurrence-free response of lacrimal gland and choroidal lesions as well as parotid granulomas and pulmonary infiltrates after 3 months of minocycline therapy. Miyazaki and colleagues [68] reported the regression of nodular lesions of muscular sarcoidosis during minocycline treatment with decreased levels of angiotensin-converting enzyme, lysozyme, and soluble interleukin-2 receptor. The effectiveness of minocycline for muscular sarcoidosis in this case was confirmed when a prompt response to the minocycline

Based on the studies described in this section, the antimicrobial properties of tetracyclines are effective for treating sarcoidosis. Many researchers, have questioned the antimicrobial role of tetracyclines because tetracyclines also have anti-inflammatory properties, which were demonstrated by in vitro studies and corroborated by clinical trials. Tetracycline suppresses neutrophil migration and chemotaxis [69], and minocycline inhibits T-lymphocyte activation and proliferation [70]. Both minocycline and doxycycline obviate granuloma formation in vitro [71]. Although it remains controversial whether these antibiotics kill microbes or have only an anti-inflammatory effect, the mechanisms of sarcoid granuloma formation caused by *P.*

help to improve the treatment of sarcoidosis.

**9.2. Tetracyclines for treating sarcoidosis**

pulmonary involvement.

28 Sarcoidosis

therapy was repeatedly observed.

The Marshall protocol [72] is an eradication therapy for intracellular bacteria established by Dr. Trevor Marshall. This therapeutic protocol is a combination of minocycline plus azithro‐ mycin or clindamycin, supported by the use of an angiotensin receptor blocker to prevent Herxheimer reactions. According to the results published by the Autoimmunity Research Foundation in 2006, this therapy is effective in 62% of patients with sarcoidosis. Information about the treatment can be found at the study site, marshallprotocol.com and also at autoim‐ munityresearch.org.

### **Author details**

Yoshinobu Eishi\*

Address all correspondence to: eishi.path@tmd.ac.jp

Department of Human Pathology, Division of Surgical Pathology, Tokyo Medical and Den‐ tal University, Japan

### **References**


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**Chapter 2**

**The Role of Type I IFN and**

Additional information is available at the end of the chapter

Mitsuteru Akahoshi

**1. Introduction**

the sarcoidosis group [3].

http://dx.doi.org/10.5772/55555

**TNF-α in the Pathogenesis of Sarcoidosis**

Sarcoidosis is a chronic systemic disorder of an unknown etiology and it is characterized by the presence of noncaseating granulomas in multiple organs. The granulomatous leison affected by sarcoidosis is marked by the accumulation and activation of CD4+ helper T (Th) cells with the Th1 phenotype and monocytes/macrophages, which suggest that a Th1-type immune response plays a dominant role in the disease pathogenesis [1]. For example, the important roles for IFN-γ and IL-12 were found in sarcoid lung [2], and a genome-wide gene expression analysis of sarcoid lund tissues identified signal transducer and activator of transcription-1 gene (*STAT1*) as one of the dominant network genes most highly expressed in

IFN-α is known to be a potent stimulator of Th1 immune response, and increased type I IFN signaling has been implicated in a number of autoimmune diseases such as systemic lupus erythematosus (SLE) [4]. On the other hand, type I IFN has been used to treat a variety of diseases, including chronic hepatitis C (HCV) infection. However, due to its immunomedu‐ latory effects, it has also been reported to induce several autoimmune and/or inflammatory disorders [5]. Notably, an increasing number of sarcoidosis has been reported in chronic HCV patients who received type I IFN therapy [6, 7]. In some cases, the sarcoid lesions improved following dose reduction or cessation of the therapy, suggesting the importance of type I IFN in the disease development. Moreover, from a genetic standpoint, we recently showed an association between polymorphisms in the *IFNA* gene and susceptibility to sarcoidosis [8]. Another recent study found that in an European-American population, serum type I IFN activity was higher in sarcodosis cases as compared to matched controls [9]. In addition, besides IFN-induced sarcoidosis, a dozen case reports of sarcidosis have also been reported in patients treated with tumor necrosis factor (TNF) antagonists [10]. A cross-regulation between

> © 2013 Akahoshi; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.


### **Chapter 2**
