Correlation Between TLR-2 Level and Clinical or Radiological Severity on Patients

with Tuberculosis Spondylitis in Dr. Wahidin Sudirohusodo Hospital Makassar

Jainal Arifin1, M. Ruksal Saleh1, Risang Setyo Argo1*, Dave Kennedy2, M. Andry Usman1

1
Department of Orthopaedic and Traumatology, Faculty of Medicine, University of Hasanuddin, Makassar,
Indonesia
2
Faculty of Medicine, University of Hasanuddin, Makassar, Indonesia

ABSTRACT

Background:
Spinal tuberculosis or TB spondylitis is one of the most common types of extra pulmonary tuberculosis, which
is about 15% of all cases. It causes severe morbidity, neurological deficits, and severe deformities on the
patients. The growth of Mycobacterium tuberculosis in culture specimens obtained from infected tissue is the
single gold standard diagnostic test for spinal TB. Toll-like receptor (TLR) is one of the important receptors in
the first-line defence system against microbes. TLR-2 and TLR-4 are known to be associated with tuberculosis
infection. Based on this background, the researchers were interested in examining the relationship between
TLR-2 levels and the clinical and radiological severity of TB spondylitis patients.
Methods: A cross-sectional study was conducted with patients diagnosed with tuberculosis spondylitis at Dr.
Wahidin Sudirohusodo Makassar. Patients diagnosed with TB spondylitis confirmed by blood tests,
GeneExpert, and Magnetic Resonance Imaging without prior treatment were included. Data analysis were
conducted by using descriptive analysis and One-way ANOVA for bivariate analysis.
Results: The mean value of TLR2 levels in TB spondylitis patients was 9.1 g/dL. TLR2 levels in paraesthesia
were significantly higher than normal (p <0.05) the table above results show that there is a significant
relationship between TLR2 levels and sensory neurologic status. Similar trend were analyzed on the motor
neurologic status with TLR2 levels in paraparesis were significantly higher than those in normal (p<0.05).
Conclusion: There is a significant relationship between the TLR2 levels in TB spondylitis and their impaired
motor and sensory function. Spinal destruction has been shown to provide significant relationship with TLR2
value in spondylitis TB.

Keywords: Tuberculosis; spondylitis; Toll-like receptor; TLR2

1. INTRODUCTION Asia having the second highest prevalence,

In 2017, around 10 million people which is around 226 cases per 100,000
were reported to be infected with population. Spinal tuberculosis is one of
tuberculosis worldwide, with Southeast the most common types of extra

1
pulmonary tuberculosis, which is about
15% of all cases. Infection of the spinal
cord usually causes neurological
symptoms such as pain, weakness and
cramps in the lower extremities. If left
untreated, spinal tuberculosis has severe
morbidity, neurological deficits, and
severe deformities. All of these conditions
cause impairment of function, particularly
of mobility, leading to difficulties with
walking, self-care, and sphincter function.
This can cause physical, mental, emotional
and social disturbances. (Yong et al, 2021)
The spine is the osseous part most
often infected by tuberculosis.
Tuberculous spondylitis is caused by
Mycobacterium tuberculosis, which is an
acid-fast, aerobic bacillus with slow
growth. Tuberculosis bacilli can remain
dormant for a long time. This bacterial
infection causes a granulomatous
inflammatory reaction, which is
characterized by the presence of caseous
necrosis, lymphocytes, epithelioid cells,
and Langhan’s cells. (Sae-Jung et al, 2019)
The growth of Mycobacterium
tuberculosis in culture specimens obtained
from infected tissue is the single gold
standard diagnostic test for spinal TB.
However, because of its very poor
sensitivity, histopathological studies
showing classic granulomas and staining
of smears to identify acid-fast bacilli
(AFB) are considered as references for all
other diagnostic modalities. Apart from
indirect inflammatory serological markers,
immunological tests have also been used
with varying results. Toll-like receptor is
one of the important receptors in the first-
line defence system against microbes.
These receptors can recognize invading
pathogens and release harmful molecules
from dead cells and damaged tissues and
play an important role in linking the innate
and adaptive immune systems. 5 Research
on TLRs sheds new light on the
immunopathogenesis of the infectious
disease tuberculosis. Toll-like receptors
can recognize various ligands by the
lipoprotein structure of the bacilli. Toll-
like receptors 2 and TLR-4 are known to
be associated with tuberculosis infection.
Based on this background, the researchers
were interested in examining the
relationship between TLR-2 levels and the
clinical and radiological severity of TB
spondylitis patients. (Somvanshi et al,
2011)
2. MATERIAL AND METHODS
This study was approved by
medical ethics committee of Hasanuddin
University of Makassar and included
patients diagnosed with tuberculosis
spondylitis at Dr. Wahidin Sudirohusodo
Makassar. Samples were taken using the
consecutive sampling technique method.
The data taken is primary data that was
2
examined in TB Spondylitis patients who these patients was found to be around
visited in the period July-December 2022. 66.7%.
Statistical data analysis and data
distribution used the Kruskall Wallis and As shown in Table 1 we found that spinal
Spearman alternative One-Way ANOVA cord destruction less than 3 and 33.3%
method. more than 3. On assessment of
The inclusion criteria involved neurological function, motor disturbances
patients with TB Spondylitis through were found in 18.5% of patients with
anamnesis, physical examination and paraparesis and 29.6% of patients with
confirmed by routine blood tests, MRI and paraplegia. In addition, for sensory
GeneExpert with the range of age between function, approximately 40.7% of patients
10-50 years old and no administration of experienced hypoesthesia and 14.8% of
TB treatment whatsoever. The exclusion patients experienced anaesthesia. TLR2
criteria included patients that refused to levels in these patients had a mean value
participate and incomplete sample data of 9.1±8.07 (p<0.05). The full
while also patients with confirmed characteristics of samples are found in
extrapulmonary TB other than spine, Table 2 to Table 7
without or with tuberculosis treatment. We
have also excluded patients with Distribution of data on 27 participants
malignancy, cardiovascular disease, type 1 using the Shapiro Milk test showed that
and 2 diabetes mellitus and autoimmune the data distribution was not normal (p =
diseases, as well as immunocompromised 0.007; Figure 1). This informs that some
diseases (such as HIV-AIDS). of the distribution of participant data is
still far from the general mean value.
3. RESULTS Here's a QQQ plot for the data.
Based on the data collected, several
results were obtained related to research Figure 1. QQ plot of study participant
participants such as age, sex, vertebrae distribution based on TLR2.
damage assessed from X-Ray and MRI,
neurological disorders and TLR 2 values. 4. DISCUSSION
Participants in this study consisted of 27 Toll Like Receptors-2 (TLR2) is a
people with an average age of <50 years, major innate immune receptor that
22 people and >50 years as many as 5 recognizes various ligands present in
people (35 ± 11.8). Spinal destruction in pathogens and also serves as an
3
 13
endogenous “alarmin”. Interactions Extrapulmonary TB (EPTB). 16 In vivo, it
between TLRs and ligands present in was found that TLR2 also plays a role in
pathogens trigger intracellular signalling inducing bone loss through inflammatory
via the adapter protein MyD88 and/or processes, by inducing macrophage-
Toll/ IL-1R which in turn induces evoked osteoclastogenesis in vitro in
17
Interferon-β (IFN-β), thereby causing response to bacteria. The inflammatory
rapid activation of the acute inflammatory response also triggers TNF production.
response in macrophages. In addition, Furthermore, excessive TNF production is
acute macrophage activation is also associated with various inflammatory
mediated by inflammatory cytokine disorders and is involved in the
receptors such as Tumour Necrosis Factor- acceleration of bone metabolism which
Receptor (TNFR). TNFR engagement also plays a role in bone loss, 1 as occurs in
activates the transcription factor NF-κB STB. 18
and Mitogen Activated Protein Kinase
(MAPK), and triggers the expression of Correlation between TLR2 and
proinflammatory cytokines, chemokines, vertebrae destruction
and microbicide molecules. Although In our study we found a significant
important for the immune response. 14 association between spinal damage
Mycobacterium tuberculosis (p<0.005). This is in accordance with
(MTB) expressing TLR2 ligand. There are several sources who say that there are
several MTB ligands which are TLR2 several aetiologies that can cause damage
agonists, namely Lipoprotein 19kDa to the spine, one of which is associated
(LpqH). Mycobacterial lipoproteins, LprA with the role of TLR2 in triggering bone
(Rv1270), LprG (Rv1411c) and PhoS1. In damage. Based on the results of research
general, it can be assumed that TLR2 obtained from Nature research published
triggers three functional responses in MTB in the National Library of Medicine in
infection which include protection, 2016 explaining that the role of TLR2 and
avoidance, and MTB regulation of the TLR4 is to assist the synaptic process of
immune system. This mechanism does not TREM-2 to regulate IL-1B and IL-6 in the
really play a role in acute infection but initiation of secretion from MMP-2 and
plays a role in chronic infection. 15 MMP -8 and osteoclast activation in
TB spondylitis (STB) is one of the experiments on cholesteatoma which
manifestations of MTB infection and induces an inflammatory process and then
contributes to around 10% of
4
triggers the process of destruction of a
bone. 19
Then in a further study reported
from Immunology Letters in 2007
explained that Toll-Like Receptors (TLRs)
in this case TLR2 and TLR4 induce
tartrate-resistant acid phosphatase (TRAP)
and RANKL in Rheumathoid Arthritis
conditions thereby triggering osteoclasts to
undergo a process of differentiation. Thus,
the study concluded that targeting therapy
at specific TLRs might be an approach in
preventing inflammation that can trigger
bone destruction in the pathogenesis of
rheumatoid arthritis. 20
In other experimental studies
also reported about how the role of
Porphyromonas gingivalis as the primary
aetiology of periodontal infection which
can develop into loss of alveolar bone
density. In a 2013 study in The Journal of
Immunology, it was reported about the
role of TLR2 and TLR4 which mediate the
process of P. gingivalis in producing
cytokines to activate TNF to create an
inflammatory state which then progresses
to the process of loss of bone density. 21
Based on the three reports of
previous research results, it can be
concluded that TLR2 indirectly plays a
role in the process of mediating
inflammatory mediator complexes such as
TRAP, RANKL, IL-1B, IL-6 and TNF in
conditions of acute, chronic infection or
other conditions that have high potential
for inflammation, and It then develops into
an inflammatory state that induces
destruction of a bone. 19,20,21
Correlation of TLR2 and sensory
disturbances
The study found a significant
relationship between TLR2 and sensory
disturbances in study participants
(p<0.005). It is currently thought that pain
is only mediated by neurons. However,
recent findings about the role of spinal
cord glial cells suggest a different role for
pathological pain hypersensitivity. There is
increasing evidence that glial cell activity
in the spinal cord plays an important role
in the initiation or maintenance of
pathological pain of various etiologies.
TLRs on the neuroimmune surface and
some evidence suggests that inflammation
from TLRs activates in glial cells and
affects nociceptive processes and causes a
painful and persistent state.
The mean TLR2 level was
found to be highest under anesthesia
(16.93) and lowest under normal (2.29),
while the difference in TLR2 levels based
on sensory is very significant (p<0.001)
and the results of the follow-up test (LSD)
showed that TLR2 levels in anesthesia
were also higher than those in paraesthesia
(13.72), but not statistically significant
5
(p>0.05) [Figure 2, Table 8]. TLR2 nociceptive pathways. Acute intrathecal
levels in paraesthesia were significantly delivery of TLR2 agonists induces lasting
higher than normal (p <0.05) the table tactile allodynia. In this study, mixed
above results show that there is a spinal cord glial cell cultures from wild-
significant relationship between TLR2 type mice, incubation with damaged
levels and sensory neurologic status. sensory neuron supernatants generated
many pronociceptive inflammatory genes,
Figure 2. Comparison of TLR2 levels such as TNF, IL-1b, IL-6, and iNOS. In
according to sensory contrast, glials isolated from TLR2
mutants, did not show changes in cytokine
TLRs are pattern recognition and iNOS gene expression in response to
receptors that initiate innate immune sensory neuron damage. Nerve transection
responses by recognizing the molecular injuries lead to increased TLR2
structures of various pathogens, such as immunoreactivity in the spinal cord
pathogen-associated molecular particles accompanied by increased microglial
(PAMPs). Following tissue injury or NADPH oxidase. 23
cellular stress, TLRs also detect
endogenous ligands known as danger- Correlation of TLR 2 and motor
associated molecular patterns (DAMPs). impairment
TLRs are expressed in non-neural and In this study, a significant
central nervous system (CNS) cell types. association was found between TLR2
Following tissue disruption and nerve levels and motor disturbances in
injury, TLRs (TLR2, TLR3, and TLR4) participants. This is supported by the fact
induce activation of microglia in astrocytes that Toll-like receptor 2 (TLR2) is one of
and production of proinflammatory the receptors that plays a role in the
cytokines in the spinal cord, leading to the immune system. TLR2 is a membrane
development of inflammatory and protein which are expressed on the surface
neuropathic pain. TLRs are novel in their of certain cells and recognize foreign
role in processing pain and itching by substances and transmit appropriate
increasing the excitability of primary signals to immune system cells.The first
sensory neurons. 22 study of the relationship between TLR2
TLR2 specifically contributes and motor disturbances revealed a role for
to persistent pain through TLR2 and TLR4 in p38 activation
neuroinflammatory mechanisms in (Mitogen Activated Protein Kinase)
6
MAPK and Jun N Terminal Kinase (JNK)
during endurance exercise in rats.
The mean TLR2 level was found
to be highest in paraplegia (18.30) and
lowest in normal (2.73). we found that the
difference in TLR2 levels based on motor
was very significant (p<0.001) which
when the of the follow-up test was
conducted (LSD) showed that TLR2 levels
in paraplegia were also significantly higher
than those in paraparesis (12.29) (p<0.05)
[Table 9, Figure 3]. It was also found that
TLR2 levels in paraparesis were
significantly higher than those in normal
(p<0.05). The above results show that
there is a significant relationship between
TLR2 levels and motor neurologic status.
Figure 3. Comparison of TLR2 levels by
motor.
Several studies have shown that
MAPK signalling can be increased in
response to muscle contraction in the
complete absence of systemic factors.
Evidence from isolated muscle preparation
studies suggests that p38 MAPK activation
can be triggered by calcium/calmodulin-
dependent protein kinase II, suggesting
that the cytosolic calcium concentration in
skeletal muscle increases during exercise.
may mediate Exercise-induced activation
of MAPK p38.1
JNK activation appears to be very
sensitive to stretching because eccentric
contractions induce greater JNK activation
compared to concentric contractions. The
results of this study reveal that the
signalling cascade initiated by TLR2 and
TLR4 has a major role in the activation of
p38 MAPK and JNK during endurance
running exercise in mice. 22,23
Enteric Nervous System (ENS) is a
complex and interconnected network of
neural and glial circuits located throughout
the digestive tract, which exerts intrinsic
control on intestinal motility and secretion.
Together with the Central Nervous
System, the ENS forms a bidirectional
ENS-CNS axis, which is also closely
related to the immune system. There is
mounting evidence that TLR2 along with
TLR4 is abundantly present in the plasma
membrane of enteric neurons and
participates in enteric neuron signalling.
TLR2 is a cell surface receptor
expressed on a variety of immune and
epithelial cells, as well as neuronal and
glial cells in the central, peripheral, and
enteric nervous systems. Activation of
TLR2 by polysaccharide A from
Bacteroides fragilis causes an anti-
inflammatory response in B cells by
producing interleukin 10 (IL-10), this
indicates the role of the TLR2
immunoregulation complex in response to
various pathogens that are often found in
7
the intestine or gut. endogenous factors
involved in neurodegeneration and cause
motor disturbances in patients with
Parkinson's disease. In conclusion, in
accordance with the findings conducted in
studies on JNK activation, TLR2 can
affect disturbances in muscle contractions
by mediating MAPK p38,22 activation and
also affect the ENS which indirectly
contributes to causing motor disturbances
in patients with Parkinson's. 3 However, it
is still needed further research into other
effects of TLR2 on motor disorders. 24
Figure 7. Correlation between vertebrae
destruction and TLR2.
The figure above shows the
correlation between vertebral destruction
and TLR2 levels. The results of the
Spearman correlation test, found that there
was a very significant positive correlation
with vertebrae destruction and TLR2
levels (p<0.001) [Figure 4], where the
more vertebrae destruction the higher the
TLR2 levels. The magnitude of the
correlation between vertebral destruction
and TLR2 levels is included in the very
strong category (R> 0.750).
The proportion of paraplegia and
paraparesis in the destruction of vertebrae
4-6 (66.7% and 33.3%) was found to be
the highest compared to the destruction of
the vertebrae <4, while the normal motor
was found to be the highest in the
destruction of the vertebrae 1 (90.9%) and
0 ( 0%) in destruction of vertebrae 4-6.
This difference was statistically significant
(p<0.01), indicating a significant
association between vertebrae destruction
and motor neurologic status (Table 10).
The proportion of anesthesia and
paraesthesia in the destruction of vertebrae
4-6 (33.3% and 66.7%) was found to be
the highest compared to the destruction of
the vertebrae <4, while normal sensory
was found to be the highest in the
destruction of the vertebrae 1 (81.8%) and
0 ( 0%) in destruction of vertebrae 4-6.
This difference was statistically significant
(p<0.01), indicating a significant
association between vertebrae destruction
and sensory neurologic status (Table 11).
5. CONCLUSIONS
We found that there was a significant
increase in TLR2 levels in patients with
TB Spondylitis with a mean value of 9.1.
There is also significant relationship
between the TLR2 levels in TB spondylitis
and their impaired motor and sensory
function. Patients and spinal destruction
has been shown to provide significant
relationship in the levels of TLR2 in
spondylitis TB
Acknowledgments
None.
8
 All named authors hereby declare that they
Conflict of interest have no conflicts of interest to disclose.

9
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