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\begin{document}

\title{M. tuberculosis infection in diabetics is associated with increased
inflammatory cytokine but decreased Suppressor of cytokine signaling
(SOCS)-3 responses}



\author[1]{Kiran Iqbal}%
\author[1]{Muhammad Irfan}%
\author[1]{Qamar Masood}%
\author[1]{Maliha Yameen}%
\author[1]{Nanik Ram}%
\author[1]{Bushra Jamil}%
\author[1]{Shoaib Rao}%
\author[2]{Martin Rottenberg}%
\author[3]{Zahra Hasan}%
\affil[1]{Aga Khan University}%
\affil[2]{Karolinska Institutet}%
\affil[3]{The Aga Khan University}%


\vspace{-1em}



  
  \date{May 05, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
Introduction Tuberculosis (TB) infections and latent Mycobacterium
tuberculosis (MTB) infection (LTBi) remain prevalent globally. Type 2
diabetes mellitus (DM) worsens TB outcomes but the immune mechanisms
that cause this are not yet clear. We investigated a role of suppressor
of cytokine signaling molecules (SOCS1 and SOC3) in regulating host
cytokine responses in the diabetic host infected with MTB. Materials and
Methods We studied peripheral blood cells from health endemic controls
(EC), LTBi cases, diabetics with and without LTBi and TB patients.
Mycobacterial antigen-stimulated cytokine secretion was determined using
the Th1/Th2 11 plex cytokine assay. Antigen-induced gene expression of
IFN\selectlanguage{greek}γ, \selectlanguage{english}TNF\selectlanguage{greek}α, \selectlanguage{english}IL6 and SOCS3 was determined by reverse-transcription PCR.
Results Purified protein derivative (PPD) antigen stimulation induced
higher levels of, IL-6, IL-2, TNF\selectlanguage{greek}α \selectlanguage{english}and GM-CSF levels in DM-LTB as
compared with EC and LTB cases. IL-13 levels were raised in DM-LTB cases
as compared with DM cases. PPD-induced IFN\selectlanguage{greek}γ \selectlanguage{english}and IL-6 transcripts were
raised in DM-LTBi as compared with EC. TNF\selectlanguage{greek}α \selectlanguage{english}mRNA levels were raised in
DM-LTBi as compared with LTBi. SOCS3 mRNA levels were reduced in DM-LTBi
as compared with LTBi. SOCS3 transcripts were higher in LTBi as compared
with EC and TB groups. Discussion We found co-occurrence of LTBi with DM
to be associated with an increased release of proinflammatory IL-6, IL-2
and TNF-\selectlanguage{greek}α \selectlanguage{english}but reduced SOCS3 mRNA levels. SOCS3 protects against MTB
infection therefore, reduced levels in DM-LTB may be contribute to
progression from LTBi to active TB in individuals infected with MTB.%
\end{abstract}%



\sloppy


\emph{M. tuberculosis} \textbf{infection in diabetics is associated with
increased inflammatory cytokine and decreased Suppressor of cytokine
signaling (SOCS)-3 responses}

Kiran Iqbal Masood\textsuperscript{1}, Muhammad
Irfan\textsuperscript{2}, Qamar Masood\textsuperscript{2}, Maliha
Yameen\textsuperscript{1}, Bushra Jamil\textsuperscript{2}, Nanik
Ram\textsuperscript{2}, Shoaib Rao\textsuperscript{1}, Martin
Rottenberg\textsuperscript{3}, Zahra Hasan\textsuperscript{1}*

\textsuperscript{1}Department of Pathology and Laboratory Medicine, The
Aga Khan University Karachi, Pakistan; \textsuperscript{2}Department of
Medicine, The Aga Khan University Karachi,
Pakistan;\textsuperscript{3}Department of Microbiology, Tumor and Cell
Biology, Karolinska Institutet, Sweden.

Running Title: Increased inflammation in diabetics with latent TB is
associated with reduced SOCS3 gene expression

Scientific heading: Clinical and Human Immunology

*Corresponding Author:

Zahra Hasan, PhD, FRCPath

Professor

Department of Pathology and Laboratory Medicine

The Aga Khan University

Stadium Road, P.O.Box 3500

Karachi 74800, Pakistan

Email: zahra.hasan@aku.edu

Contact: +92 21 3486 4527

Word count: 5079

\textbf{Summary}

\textbf{Introduction}

Tuberculosis (TB) infections and latent \emph{Mycobacterium
tuberculosis} (MTB) infection (LTBi) remain prevalent globally. Type 2
diabetes mellitus (DM) worsens TB outcomes but the immune mechanisms
that causes this are not yet clear. We investigated a role of suppressor
of cytokine signaling molecules (SOCS1 and SOC3) in regulating host
cytokine responses in the diabetic host infected with MTB.

\textbf{Materials and Methods}

We studied peripheral blood cells from health endemic controls (EC),
LTBi cases, diabetics with and without LTBi and TB patients.
Mycobacterial antigen-stimulated cytokine secretion was determined using
the Th1/Th2 11 plex cytokine assay. Antigen-induced gene expression of
IFN\selectlanguage{greek}γ, \selectlanguage{english}TNF\selectlanguage{greek}α, \selectlanguage{english}IL6 and SOCS3 was determined by reverse-transcription PCR.

\textbf{Results}

Purified protein derivative (PPD) antigen stimulation induced higher
levels of, IL-6, IL-2, TNF\selectlanguage{greek}α \selectlanguage{english}and GM-CSF levels in DM-LTB as compared with
EC and LTB cases. IL-13 levels were raised in DM-LTB cases as compared
with DM cases. PPD-induced IFN\selectlanguage{greek}γ \selectlanguage{english}and IL-6 transcripts were raised in
DM-LTBi as compared with EC. TNF\selectlanguage{greek}α \selectlanguage{english}mRNA levels were raised in DM-LTBi as
compared with LTBi. SOCS3 mRNA levels were reduced in DM-LTBi as
compared with LTBi. SOCS3 transcripts were higher in LTBi as compared
with EC and TB groups.

\textbf{Discussion}

We found co-occurrence of LTBi with DM to be associated with an
increased release of proinflammatory IL-6, IL-2 and TNF-\selectlanguage{greek}α \selectlanguage{english}but reduced
SOCS3 mRNA levels. SOCS3 protects against MTB infection therefore,
reduced levels in DM-LTB may be contribute to progression from LTBi to
active TB in individuals infected with MTB.

Key words: MTB, latent TB, diabetes, SOCS3

\textbf{Introduction}

One third of the people suffering with type 2 diabetes mellitus (DM)
reside in developing countries many of which are endemic for
Tuberculosis (TB). In Pakistan, the prevalence of diabetes is reported
to be 9.8\% and 3\% of all national deaths are thought to be associated
with DM and DM-related conditions (1). As Pakistan also ranks
5\textsuperscript{th} amongst high TB burden countries, it is an
additional challenge to manage patients with TB and DM comorbidity (2).
Different risk factors may account for an important proportion of TB
cases in the population. Diabetes (DM) occurs as the individual becomes
resistant to insulin, resulting in poor metabolism of glucose.
Individuals with DM have a threefold increased risk of developing TB (3,
4). DM has also a negative impact on treatment outcomes in patients with
tuberculosis (TB) (5). Latent, asymptomatic infection or immune
reactivity with \emph{M. tuberculosis} (LTBi) occurs in around one
fourth of the world population (6), and the association between LTBi and
DM-2 has been suggested (7).

Studies on host immunity of individuals with LTBi indicate that CD4 T
cell responses specific to MTB antigens are raised in this group (8).
Which is the premise of the MTB-specific T cell -derived IFN\selectlanguage{greek}γ \selectlanguage{english}assay
testing for LTBi diagnosis (9). Additional cytokines and chemokines such
as IL-10, TNF\selectlanguage{greek}α, \selectlanguage{english}CXCL10, CXCL9 are also found to be raised in LTBi as
compared with healthy endemic controls (EC) (10). Gene expression
studies have identified particular biomarker expression patterns within
LTBi individuals that discriminate them from those who are healthy and
those who have active TB (11) (12). Heterogeneity of LTBI was further
recognised by epidemiological differences in the risk of TB between
those with recent and remote infection (13). It is well understood that
immune regulatory factors such as, IL-12, IFN-\selectlanguage{greek}γ \selectlanguage{english}and TNF-\selectlanguage{greek}α \selectlanguage{english}confer
protective immunity against MTB infection, the understanding of factors
which lead to disease progression from LTBi to TB is very limited (14).

DM is associated with altered cellular and humoral immune responses with
modulation of macrophage and lymphocyte function (15). Delayed
management of DM can lead to diverse complications including
susceptibility to infections (16). Inflammatory mediators including,
proinflammatory cytokines such as IFN\selectlanguage{greek}γ, \selectlanguage{english}TNF\selectlanguage{greek}α, \selectlanguage{english}IL-6, and downregulatory
cytokines IL-10, TGF\selectlanguage{greek}β \selectlanguage{english}together with adipokines, which affect leptin
regulation and impact metabolism in glycogen stores, are all raised in
DM cases (17). Individuals with LTBi and DM have been shown to
sub-optimal CD4 T cell responses to MTB-specific antigens and increased
production of the Th 2 cytokines IL-10 and TGF-beta (18).

Host innate and adaptive responses are both crucial in protection
against TB. Suppressor of cytokines signaling (SOCS) molecules regulate
key cytokine drivers of activation of both innate and adaptive immunity.
SOCS has eight member molecules including cytokine-inducible regulator
of signaling (CIS), SOCS1-7 (19). SOCS1 and 3 are found to play a role
in the pathogenesis of TB and diabetes (20). SOCS1 suppresses STAT1, the
transcription factor involved in IFN-\selectlanguage{greek}γ \selectlanguage{english}and IFN-\selectlanguage{greek}α/β \selectlanguage{english}signaling, while
SOSC3 has different targets, including STAT3 activation via certain
receptors as the IL6-receptor family (21). SOCS1 and 3 molecules are
modulated by MTB for its own survival, play a pivotal role in
controlling infection outcomes (19, 22, 23).

Separately, SOCS1 and SOCS3 inhibit the insulin receptor substrates 1
and -2 (IRS-1 and IRS-2) protein, hindering insulin responses and
insulin growth factor (IGF)-1 signaling, thereby affecting glucose
metabolism (24). Here we investigated the impact of diabetes on
progression of MTB infection to active disease through the study of
mycobacterial antigen-induced cytokine activation and their association
of regulatory SOCS1 and SOCS3.

\textbf{Materials and Methods}

\textbf{Ethical approval}

This study was approved by the Ethical Review Committee of The Aga Khan
University Karachi, Pakistan. Written informed consent was taken from
all study participants.

\textbf{Subject selection}

Study subjects were recruited from Aga Khan University and Hospital
(AKUH), Karachi, Pakistan using a cross-sectional study design with a
consecutive convenience sampling method. Study subjects were recruited
within a given study period based on the given inclusion criteria.

All study subjects were aged 18 -- 65 years. Thirty-nine asymptomatic
healthy volunteers at AKUH were recruited for the study. We recruited
health care workers who have contact with TB patients as possible LTB
cases in addition to household contacts of TB patients. Thirty-three
patients with DM were recruited from Department of Medicine clinics.
Inclusion criteria for DM cases were: patients with previously confirmed
Type 2 DM. Exclusion criteria were pregnancy, comorbid conditions such
as HIV infection, active TB, chronic renal failure, chronic liver
disease or corticosteroid therapy. For potential DM-LTBi cases we
recruited household contacts of TB patients who had DM.

In total, ten ml of whole blood sample was drawn from all study
subjects. Random blood glucose (RBS) and glycosylated hemoglobin (HbA1c)
levels were tested. DM was defined by HbA1c [?] 6.4 \%.

A positive reaction in a Quantiferon TB Gold in Tube assay (QFT-GIT)
test was performed to classify individuals which are otherwise are
healthy and asymptomatic as LTBi. As per manufacturer's instructions, a
threshold of [?]0.35 IU/mL was considered for LTBi. Hence after QFT-GIT
testing, the healthy control group was categorized into endemic controls
(EC n=24) or latent TB cases (LTBi, n=15). Further, subjects recruited
in the DM group were further categorized into QFT-GIT negative (DM,
n=16) and positive (DM-LTBi, n=17).

Fifteen patients with pulmonary TB (PTB) were recruited. Patients with
confirmed diagnosis of TB who had not received anti-tuberculous therapy
(ATT); male or female; between 18--65 years of age were included in the
study. Exclusion criteria were pregnancy, comorbid conditions (such as
HIV infection, DM, chronic renal failure, chronic liver disease or
corticosteroid therapy) and patients with relapsed TB. All PTB patients
were diagnosed by clinical examination, chest X-ray and had a positive
sputum acid-fast bacillus (AFB) microscopy and/ or AFB culture. They
were classified as PTB as per WHO guidelines (25).

\textbf{Stimulation of PBMCs with mycobacterial antigens}

Five ml of blood was used for isolation of peripheral blood mononuclear
cells (PBMCs). Briefly, PBMCs were separated using Ficoll-Histopaque as
described (26) and plated at 10\textsuperscript{6} cells/ well in a 24
well plate. Cells were incubated with purified protein derivative
antigen (PPD) at 10 \selectlanguage{greek}µ\selectlanguage{english}g/ml (BeiResources, Colorado State University, USA)
or left unstimulated for 18h at 37\textsuperscript{o}C. Cellular
supernatants were collected after centrifugation. Cells were washed and
the pellets were snap-frozen at -80\selectlanguage{ngerman}°C until tested. Cell monolayers were
lysed and harvested using TriZol reagent (Invitrogen, USA) and stored at
-70°C until processed further.

\selectlanguage{english}\textbf{Measurement of cytokines by Th1/Th2 Cytokine 11-Plex Human
ProcartaPlex assay}

Cytokines were measured in serum collected from whole blood. Also, in
cellular supernatants of PBMC monolayers after 18 h of incubation with
and without PPD antigen stimulation. Concentrations of IFN-\selectlanguage{greek}γ, \selectlanguage{english}IL-12,
IL-2, TNF-\selectlanguage{greek}α, \selectlanguage{english}GM-CSF, IL-1, IL-13, IL-4, IL-5, IL-6 and IL-18 were
measured in cell supernatants with a Th1/Th2 Cytokine 11-Plex Human
ProcartaPlex assay from ThermoFischer Scientific, USA. Readings were
taken on a Luminex 100 instrument (Luminex technologies, USA).

\textbf{Real Time PCR}

Total RNA was isolated from PBMCs using Trizol reagent. RNA (1 \selectlanguage{greek}μ\selectlanguage{english}g) was
reverse transcribed using RevertAid First Strand cDNA Synthesis Kit
(Thermo Fisher Scientific, USA) as described by the manufacturer. Real
time PCR was performed in duplicate 20 \selectlanguage{greek}μ\selectlanguage{english}l reactions containing Platinum
SYBR Green qPCR Supermix-UDG (Thermofisher scientific, USA), 150 nM
forward and reverse primers, and 2 \selectlanguage{greek}μ\selectlanguage{english}l of cDNA. HuPO (human acidic
ribosomal protein) primer sequences were obtained from published reports
as described previously (27). IFN-\selectlanguage{greek}γ, \selectlanguage{english}TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-6, SOCS1 and SOCS3
sequence specific primers were used. Two-fold dilutions of cDNA samples
were amplified to control amplification efficiency and to determine the
optimal concentration required for each primer pair. HuPO was used as a
control gene to calculate the \selectlanguage{greek}Δ\selectlanguage{english}Ct values for individual samples. The
relative amount of cytokine/HuPO transcripts was calculated using the
2\textsuperscript{-[?][?]cT} method as described (28). These values were
then used to calculate the relative expression of cytokine mRNA in each
of the samples tested.

\textbf{Statistical analysis}

Data is depicted as median values for each group with the IQR (inter
quartile range 25th to 75th percentile) indicated in each case.
Comparison of non-parametric data between the groups was performed using
the Mann--Whitney U test. Analysis was performed and data plotted using
GraphPad PRISM Version 5 (GraphPad Software, San Diego, CA, USA).

\textbf{Results}

\textbf{Characteristics of study subjects}

The age (mean \selectlanguage{ngerman}± SD; EC, 41.3± 11.1 y; DM, 50.4 ± 7.77 y) and gender
distribution (Male/Female; EC, 18/21; DM, 20/13) of EC and DM study
subjects was comparable (Table 1).

Patients with TB had a mean age of 40 y and comprised 11: 4 Male/Female
cases. We used the QuantiFERON-gold In-Tube assay (QFT-GIT) to determine
the latent TB status of each case and found that 15 of 39 healthy
controls and 17 of 33 DM cases had LTBi.

\textbf{Mycobacterial antigen-induced pro-inflammatory cytokines
upregulated in DM-LTB cases}

To investigate the effect of latent MTB infection on host immunity, we
compared the levels of proinflammatory cytokines in the supernatants
from purified protein derivative (PPD)- stimulated healthy and diabetic
individuals with and without LTBi. Specifically, IFN\selectlanguage{greek}γ, \selectlanguage{english}IL-6, IL-2,
IL-12, TNF-\selectlanguage{greek}α, \selectlanguage{english}GM-CSF, IL-1\selectlanguage{greek}β \selectlanguage{english}and IL-18 levels were measured in
supernatants of PPD-stimulated PBMCs in EC, LTBi, DM and DM-LTB groups.

PPD-stimulated IL-6 (p=0.011), IL-2 (p=0.0131), TNF-\selectlanguage{greek}α \selectlanguage{english}(p=0.027) and
GM-CSF (p=0.0075) levels were found to be significantly different
between the study groups, Table 2. These cytokines were found to be
raised in DM-LTB study subjects as compared with EC and/or LTBi and DM
groups. IL-6 levels were significantly raised in DM-LTBi as compared
with EC (p=0.0015) and LTB (p=0.0142). DM-LTBi cases had raised IL-2
levels as compared with EC (p.\selectlanguage{english}0.0015), LTBi (p=0.0187) and DM (p=0.0388)
cases. TNF-\selectlanguage{greek}α \selectlanguage{english}levels were raised in DM-LTBi as compared with EC
(p=0.0098), LTBi (p=0.0141) and DM (p=0.0118) cases. In DM-LTBi cases,
GM-CSF levels were significantly raised as compared with EC (p=0.0014),
LTBi (p=0.0394). GM-CSF levels were also raised in TB as compared with
EC (p=0.0075) and LTBi (p=0.038). IL-1\selectlanguage{greek}β \selectlanguage{english}levels were significantly raised
in DM-LTB as compared with EC (p=0.0020). IL-18 levels were increased in
DM-LTB as compared with EC (p=0.0090).

We observed that levels of IFN-\selectlanguage{greek}γ \selectlanguage{english}showed an increased trend in DM-LTB as
compared with EC, DM and LTB groups however, these were not
significantly different.

\textbf{Mycobacterial antigen-induced IL-13 and IL-5 levels are raised
in DM-LTB cases}

To investigate the effect of mycobacterial antigens on type 2 cytokines
we measured PPD-induced secretion of IL-13, IL-5 and IL-4 in DM and EC
cases with and without LTBi. PPD- stimulated IL-5 (p=0.025) levels were
found to be differ between study groups (Table 3). PPD-stimulated IL-5
levels were also raised in DM-LTBi as compared with EC (p=0.0288). IL-13
levels were significantly increased in DM-LTB as compared with DM
(p=0.0093). IL-4 secretion was not detected in the supernatants studied.

\selectlanguage{greek}\textbf{Differential IFN-γ, TNF-α, IL-6 and SOCS3 mRNA expression levels
in healthy controls and diabetics with and without latent TB}\selectlanguage{english}

To understand cytokine activation in our study subjects in the context
of immune-regulatory molecules, we compared gene transcription levels
and the key regulatory molecules SOCS1 and SOCS3 in healthy controls and
diabetics to investigate any effect of latent TB infection.

We measured determined PPD- stimulated mRNA levels in peripheral blood
cells from EC, LTBi, DM, DM-LTBi and TB groups. We observed increased
IFN-\selectlanguage{greek}γ \selectlanguage{english}mRNA titers in DM-LTBi (p=0.033) and TB (p=0.046) as compared with
EC (Fig 1A). PPD-stimulated TNF\selectlanguage{greek}α \selectlanguage{english}mRNA expression was increased in DM-LTB
as compared with EC (p=0.035, Fig. 1B). PPD-stimulated IL-6 (p=0.009,
Fig 1C) mRNA expression was increased in DM-LTBi as compared with EC.

SOCS3 mRNA expression was found to be significantly reduced in
PPD-stimulated cells of DM-LTBi (p=0.0422), TB (p=0.019) and EC
(p=0.025) as compared with LTBi cases (Fig 1D). SOCS1 mRNA expression
level was comparable in all the study groups (data not shown).

Basal levels of mRNA transcripts for IFN-\selectlanguage{greek}γ, \selectlanguage{english}TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-6, SOCS1 and SOCS3
mRNA were determined in all groups were similar.

\textbf{Discussion}

The association between LTBi and DM-2 has implications on global TB
control: a mathematical model predicts that elimination of TB by the
year 2035 might be hampered if the incidence of DM-2 continues to
increase worldwide (29). An elevated glucose or HbA1c levels has been
shown to be related with an increased risk of developing LTBi (30).

The aim of our study is to contribute to understanding the impact of
diabetes on host immunity during mycobacterial infection and disease.
Diabetes has been shown to be associated with altered cellular and
humoral immune responses. It has been suggested that when DM co-occurs
with TB, host protective immune function is affected (31). This leads to
more un-favourable outcomes of both TB and DM (31), with a slower
recovery and increased chance of TB relapse (32). Less is known about
the alterations of host immune responses in DM in LTBi individuals.
Limited reports indicate that there is an IL-10 and TGF-beta driven
decrease in host protective CD4 T cell responses (10).

We studied PPD-triggered cytokine responses in healthy subjects with and
without LTBi, diabetics with and without LTBi and also patients with TB.
We observed increased PPD-induced IL-2, TNF-\selectlanguage{greek}α \selectlanguage{english}and IL-6 protein levels in
DM-LTBi as compared with EC, LTBi and DM. PPD- stimulated GM-CSF levels
were also raised in DM-LTBi as compared with EC and LTBi groups.

IL-5 protein titers were higher in DM-LTBi compared with EC cases and
IL-13 levels were raised in DM-LTBi as compared with DM as well,
suggesting heightened levels of some Th2 cytokines in diabetics with
latent TB. As the latter are markers of alternate macrophage activation
(11), this suggests a possible shift to macrophage dysfunction in
diabetics who are latently infected with MTB.

The increased susceptibility to \emph{M. tuberculosis} infection is
regulated by alterations in of IFN-\selectlanguage{greek}γ, \selectlanguage{english}TNF-\selectlanguage{greek}α, \selectlanguage{english}IL1-\selectlanguage{greek}β, \selectlanguage{english}IL-6 and IL-10
levels (33, 34). Studies in diabetic mice have shown reduced levels of
IFN-\selectlanguage{greek}γ \selectlanguage{english}in lungs after infection with \emph{M. tuberculosis} and increased
bacterial load as compared with the non-diabetic ones (35).

An impaired trafficking of immune cells, resulting in delayed T cell
responses was observed to the site of infection in diabetic mouse model
resulting in increased susceptibility to infection with \emph{M.
tuberculosis} (36, 37). TNF-\selectlanguage{greek}α \selectlanguage{english}is a major regulator of granuloma
formation and hence containment of \emph{M. tuberculosis} infection.
However, dysregulation in TNF-\selectlanguage{greek}α \selectlanguage{english}levels also leads to the rupture of
granulomas resulting in dissemination of granulomas (38).

We observed increased TNF-\selectlanguage{greek}α \selectlanguage{english}gene expression at the protein and mRNA
levels in DM-LTB as compared with EC and LTBi. Elevated levels of TNF-\selectlanguage{greek}α
\selectlanguage{english}have been reported in patients with chronic diabetes (39). Raised TNF\selectlanguage{greek}α
\selectlanguage{english}levels have also observed in patients with raised HbA1c levels (39).
IL-2 and GM-CSF were shown to be raised in DM-LTB as compared with EC.
These findings are in line with previous studies (40, 41). Patients with
diabetes are known to exhibit a persistent pro-inflammatory state linked
with the development of insulin resistance and pathology linked with
diabetes (42).

We found IL-6 protein levels to be significantly raised in DM-LTB cases
as compared with both EC and LTBi subjects. Similarly, we found
PPD-induced IL-6 mRNA expression to be raised in DM-LTB subjects as
compared with EC. Raised levels of IL-6 in plasma has been shown to be
associated with metabolic disorders (43). Raised IL-6 mRNA levels have
been reported in insulin resistant humans (43). In another study, a
positive association has been reported between elevated levels of IL-6
and poorly controlled diabetes (39). In TB, IL-6 has been shown to play
a vital role in pro-inflammatory responses including promoting T cell
and B cell responses (44). IL-6 deficient mice have been shown to be
susceptible to TB infection (45).

We also observed IL-5 levels to be raised in DM-LTB as compared with
healthy controls. Previously, TB with diabetes has been associated with
an increase in circulating IL-5 levels(46) . Further, PPD-induced IL-13
secretion was raised in DM-LTB cases as compared with those with DM
only. IL-13 has previously been shown to be raised in TB patients with
pre-DM (47). IL-13 regulation is required for effective glucogenesis
(48).

Our observation that individuals with PPD-induced SOCS1 was upregulated
the most in TB patients is concordant with previous reports (49).
Previously, it has been shown that \emph{M. bovis} BCG (50) and \emph{M.
tuberculosis} infection upregulates SOCS1 expression (51). We also
observed an increase in SOCS1 upregulation in diabetics. Previous
reports showed that SOCS1 inhibit insulin receptor substrate (IRS)
proteins thereby causing insulin resistance (52).

After PPD-stimulation of PBMCs, SOCS3 expression was found to be the
highest in LTBi cases and reduced in EC, DM, DM-LTBi and TB cases.
Upregulation of SOCS3 in response to \emph{M. tuberculosis} infection
has been shown previously (23). SOCS3 is required for protection against
MTB infection and mice deficient in SOCS3 are susceptible to
mycobacterial infections (53). Upregulation of SOCS3 in macrophages has
been shown to provide protection against infection by intracellular
pathogens (23). We have previously found reduced SOCS3 gene expression
in patients with far advanced PTB (54). In this case, the association of
downregulated SOCS3 together with raised inflammatory cytokines in
DM-LTBi may be suggestive of less protective immune state in the host,
making it more prone to disease progression.

This work further points to the importance of early identification of
diabetes and of latent TB both as important independent predisposing
factors for TB progression. It emphasizes the need to identify health
interventions and treatments which may aid management of the individual
prior to progression towards active TB disease.

\textbf{Author Contributions}

Conception and design: ZH and MR; Analysis and interpretation: ZH, MR,
KI, SR, MY; Drafting the manuscript for important intellectual content:
ZH, MR, KI: Patient selection: MI, QM, BJ, NR

\textbf{Acknowledgments}

This work received financial support from a VR grant, Swedish Research
Council, Sweden and the Higher Education Commission Pakistan, National
Program for Research Universities project 5218. We thank Maliha Yameen
and Amina Ajmal for assistance with patient recruitment and technical
assistance. Thanks to clinical colleagues involved in patient
recruitment

\textbf{Conflict of Interest Statement}

None declared

\textbf{References}

1. World Health Organization. Global Report on Diabetes. 2016.

2. Jeon CY, Murray MB. Diabetes mellitus increases the risk of active
tuberculosis: a systematic review of 13 observational studies. PLoS Med.
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\textbf{Table Legends}

\textbf{Table 1.} \textbf{Characteristics of study subjects.} EC,
Endemic control; LTBi, latent TB; DM, diabetes mellitus; DM-LTBi,
latently infected Diabetics; TB, tuberculosis; RBS, random blood sugar.

\textbf{Table 2. Differential pro-inflammatory cytokine secretion
between healthy controls, diabetics and LTBi cases.} EC, Endemic
control; LTBi, latent TB; DM, diabetes mellitus; DM-LTBi, latently
infected Diabetics; TB, tuberculosis. Supernatants were collected and
tested for IFN-\selectlanguage{greek}γ, \selectlanguage{english}IL-12p70, IL-2, TNF-\selectlanguage{greek}α, \selectlanguage{english}GM-CSF, and IL-1\selectlanguage{greek}β \selectlanguage{english}by Th1 Th2 11
plex procarta plex assay (Thermofischer, USA). Data is depicted as
median with IQR. Kruskal Wallis test was run between the groups to see
the difference in secretion levels and P values are included in the
table. Individual groups were also compared using Mann--Whitney U-test.
*Denotes a significant difference ([?]0.05) as compared with DM-LTBi; \#
as compared with EC; NS, not significantly different,
p\textgreater{}0.05.

\textbf{Table 3.} \textbf{Differential type 2 cytokine secretion between
healthy controls, diabetics and LTBi cases.} EC, Endemic control; LTBi,
latent TB; DM, diabetes mellitus; DM-LTBi, latently infected diabetics;
TB, tuberculosis. Supernatants were collected and tested for IL-13,
IL-4, IL-5, IL-6 and IL-18 by Th1 Th2 11 plex procarta plex assay
(Thermofischer, USA). Data is depicted as median with IQR. Kruskal
Wallis test was run between the groups to see the difference in
secretion levels and P values are included in the table. Individual
groups were also compared using Mann--Whitney U-test. *Denotes a
significant difference ([?]0.05) as compared with DM-LTBi; NS, not
significantly different, p\textgreater{}0.05.

\textbf{Figure Legend}

\textbf{Fig 1. Raised proinflammatory responses in diabetics with latent
TB.} EC, Endemic control; LTBi, latent TB; DM, diabetes mellitus;
DM-LTBi, latently infected diabetics; TB, tuberculosis. Total cellular
RNA was extracted from PBMCs subjected to RT-PCR using sequence-specific
primers to IFN-\selectlanguage{greek}γ, \selectlanguage{english}TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-6, and SOCS-3. All mRNA expression levels
were normalized to the housekeeping gene HuPO, and data calculated as
median Target gene / HuPO by the relative quantification 2\selectlanguage{greek}ΔΔ\selectlanguage{english}C t method.
Data is depicted as bar graphs for each group with median values
indicated by a horizontal line. *Denotes a significant difference
([?]0.05) between the groups using Mann--Whitney U-test. Figure shows A.
IFN-\selectlanguage{greek}γ, \selectlanguage{english}B. TNF-\selectlanguage{greek}α, \selectlanguage{english}C. IL-6 and D. SOCS-3.

\textbf{Hosted file}

\verb`Tables and figures_TB-DM.docx`  available at \url{https://authorea.com/users/309125/articles/440108-m-tuberculosis-infection-in-diabetics-is-associated-with-increased-inflammatory-cytokine-but-decreased-suppressor-of-cytokine-signaling-socs-3-responses}

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