The Determination of the Minimum Safe Level of Interleukin 10IL—10 in the Treatment of Tuberculosis when Applying IL—10 Inhibitor

ChangTai. Li

【Background】 Tuberculosis（TB）， the ninth leading cause of death over the world， is caused by Mycobacterium tuberculosis（Mtb）. In 2016， the WHO estimated that 10.4 million infected with Mtb developed visible symptoms and 1.7 million died from chronic Mtb infection. It is also estimated that about 1.8 illion people are carrying Mtb， but over 90% of the carrier will never develop to an active TB （1）. In addition， TB caused 0.4 million deaths in people co-infected with HIV in 2016， and therefore TB may account for up to 40% of HIV-related deaths annually（1）. Moreover， 25% of TB deaths cases were in children， which cause the death of 250 thousand children（1）.

【中圖分類號】 R249 【文獻(xiàn)標(biāo)志碼】A 【文章編號】1005-0019（2018）17-008-01 There were seven countries that account for 64% of latent TB infections in 2016， with India having the highest number of infections， followed by Indonesia， China， Philippines， Pakistan， Nigeria， and South Africa.In addition， over 95% of TB infections occurred in low and middle-income countries， which have 80% of the world's population（1）.In China， there were about 1 million new cases of TB infection in 2010（2）.

Mycobacterium tuberculosis is an aerobic bacteria from the family Mycobacteriace- ae that causes TB. Specially， Mtb is neither a Gram-positive bacterium nor a Gram-negative bacterium due to a unusual， waxy coating consists of mycolic acid， which can block the combination between the dye and the cell wall， on its cell surface.Surprisingly， the genome of Mtb have been sequenced in 1998， which is very early.（3）

TB is an airborne disease. Normally， people with latent TB infections（LTBI） will not transmit Mtb. However， when people with active lung TB cough， sneeze or spit， they can spread Mtb in respiratory droplets. Even a few droplets with Mtb can cause infection in an individual. TB infections do not have unique symptoms in the early stage， making diagnosis difficult. A tuberculin skin test maybe carried out to diagnosis TB， but a false positive may occur if a person has received a bacille Calmette-Guérin（BCG）vaccination. Other diagnosis methods include itronography methods（such as CT or MRI）， fiber bronchoscope inspection， BACTEC and polymerase chain reaction.

Current methods for fighting Mtb infections can be divided into prevention by vaccine， drug treatment of latent infection and treatment of symptomatic disease. For anti-tuberculosis drugs， we now have 5 first line drug and 8 second line drugs. However， drug resistant TB have arisen around the world. Vaccines will not cause drug-resistance. Nonetheless， BCG is the only licensed TB vaccine. BCG indeed have some efficacy in preventing meningeal TB and disseminated forms in children（4）. However， BCG have poor efficacy in preventing pulmonary TB， the most prevalent form of disease in adolescent and adults（5）. So， new vaccines are urgent needed.

Although there are currently 22 kinds of vaccines in clinical trials， developers are still facing challenges when developing new vaccines. A key challenge for developing successful vaccines may be due to special abilities of Mtb to suppress， inhibit， or evade immune systems. First， by infecting macrophages， Mtb can inhibit the autophage signal pathway and the fusion between the phagosome and the lysosome， thus suppressing the process that send the epitopes to the surface of cell and reduce MHC class II antigen presentation. In addition， Mtb lives in specialized bacteria endosomes instead of in the cytoplasm which inhibits MHC class I antigen presentation. Also， Mtb epitopes can be increased on the surface of uninfected cells， reduce the efficacy of antigen-specific T cells. Moreover， Mtb forms granulomas and restricts the contact between T cells and infected cells. Mtb is also able to stop the infected cells from respondingto specific cytokines such as IFN γ. However， what is most interesting is that Mtb can induce interleukin 10 expression in myeloid cells and T cells， and T cell-derived IL-10 limits control of Mtb in the lungs（6）.

HumansIL-10 is encoded by the IL10 gene（7）， which is located on chromosome 1， comprising 5 exons. IL-10 is primarily produced by monocytes and， to a lesser extent， lymphocytes， namely type 2 T helper cells （TH2）， mast cells， CD4+CD25+Foxp3+ regulatory T cells， and in a certain subset of activated T cells and B cells.IL-10 is capable of inhibiting the synthesis of pro-inflammatory cytokines such as IFN-γ， IL-2， IL-3， TNFα and GM-CSF made by cells such as macrophages and Th1 T cells which is critical in the infection of TB.IL-10 also displays a potent ability to suppress the antigen-presentation capacity of antigen presenting cells（APCs）. However， IL-10 is also stimulatory towards certain T cells （Th2）， mast cells， B cell maturation， and antibody production. In addition， IL-10's function of treating tumor was showed in tumor bearing mice for IL-10 can inhibit tumor metastasis（8）.

IL-10 controls the formationof Cyclo-oxygenase-2 （COX-2）. Lack of IL-10 has been shown to cause COX activation and resultsthromboxane receptor activation therefore causing vascular endothelial and cardiac dysfunctions in mice. IL-10 knockout frail mice develop cardiac and vascular dysfunction with increased age（9）. In addition， reduced level of IL-10 have been observed in individuals diagnosed with multiple sclerosis when compared to healthy individuals（10）.

Recombinant human IL-10 （rHuIL-10） was produced for patients who have Crohn's disease and rheumatoid arthritisreact favorably towards treatment with recombinant IL-10-producing bacteria（11）.However， rHuIL-10 treatment showed little efficiency for either Crohn's disease or rheumatoid arthritis' treatment（12）.Clinical improvement was observed in 46% （33.7-59） in the 8-microg/kg rhuIL-10 group in comparison with 27% （17-39.6） in patients taking placebo（12）.

In addition，PEGylated recombinant human IL-10 （PEG-rHuIL-10， AM0010） is now developing.A phase I clinical trial have been conducted and showed thatAM0010 led to systemic immune activation with elevated immune-stimulatory cytokines（IFNγ， IL-18， IL-7， GM-CSF and IL-4） and reduced transforming growth factor beta in the serum. Partial responses were observed in onepatient with uveal melanoma and four of 15 evaluable patients with RCC treated at 20 mg/kg （overall response rate， 27%）. Prolonged stable disease of at least 4 months was observed in four patients，including one with colorectal cancer with disease stabilization for 20 months（13）. These experiments proved that although IL-10 is immunosuppressive in bacteria-related infections， rHuIL-10/PEG-rHuIL-10 is mainly immunostimulatory in tumor treatment.

However， while the development of recombined IL-10 medicine made some progress， the development of the inhibitor of IL-10 is largely overlooked.IL-10 has beenreported to limit the protective immune response to M. tuberculosis infection， contributing to increased susceptibility to TB in a 2011 experiment（14）. Inhumans， active TB correlates with increased levels of IL-10 （15-17）. Compared with healthy controls， IL-10 has been shown to be elevated in the pleural fluid （17），bronchoalveolar lavage fluid （BALF）（16）， sputum （17）， and serum（15） of patients with active pulmonary TB （PTB）.In addition， In humans， in contrast to healthy controls， monocytes isolatedfrom PTB patients produce higher levels ofIL-10 （18）.

Moreover， this overload of IL-10 in PTB patients results in immunocompromise when fighting Mtb. It has been showed that T cell proliferationand IFN-g productionfrom BALF obtained from PTB patients have been shown to be impaired in response to Mtb stimulation by endogenousIL-10（19）. Production of IL-10 by human macrophages infected withM. tuberculosis has also been shown to inhibit phagosome maturation， resulting in impaired bacterial clearance（20）.In addition， It is showed that the blockage of IL-10 signaling when injecting BCG may enhance the efficacy of the BCG（21）.

Also， TB is not the only disease in which IL-10 suppresses the immune response to pathogens. Previous studies in 2007 and 2012 have showed that IL-10 produced by IFN-g+ Th1 cellsis downregulate the immune response against some infections， especially protozoa infections such as toxoplasmosis （22）， leishmaniasis （23）， and malaria（24）.

Based on these results， it is reasonable to speculate that an inhibitor of IL-10 may enhance immune response in Mtb infections， therefore may be a potential good adjuvant to co-implement with vaccines or anti-tuberculosis drugs.

Current Research Development

Currently， scientists have illustrated the usage of inhibiting IL-10 in TB infection. Using IL-10 reporter mice， Lúcia Moreira-Teixeiraet. al. found that both innate immune cells like monocytes， neutrophils， NK cells，and adaptive immune cells like T cells and B cells can produce IL-10 after the injection of hypervirulent W.BeijingMtbstrain HN878.The authors used WT， Il102/2， Il10fl/flLysMCre+， Il10fl/fl CD4-Cre+， Il10fl/fl CD11c-Cre+， and Il10fl/fl CD19-Cre+ mice and respective Cre2 littermate controls in the experiment. Then， the authors compared the number of Mtb in their lungs after infected with M. tuberculosis HN878 to determine whether the immune system was suppressed.

Although monocytes stand for the most portion of IL-10 production post-injection， about 70% 7 days after injection andabout 50% 21 days after injection， this study has showed that the blockage of IL-10 production in monocytes did not enhance the protection in Mtb infections， which is discovered by no significant decrease of total bacteria number. T-cells derived IL-10， especially CD4+T cells and CD8+T cells derived IL-10， is the real cause of immunosuppression in Mtb infection， because after blocked the ability of T cells to product IL-10， the number of bacteria decreased significantly（25）.

One of the major concerns about applying IL-10 inhibitor in TB treatment is the risk of causing self-immune disease such as chronic enteritis. However， an August 2017 research showed that the side effect of inhibiting IL-10 for a short period of time， such as when injecting immunization doses， may not be that serious. The authors used two different antigens， OVA and HPV16E7 long peptide as the immunogen in non-obese diabetic（DOB） mice to test the efficacy of blocking IL-10 in tumor treatment. The authors found that immunization with papilloma virus like particles， a TLR4 receptor stimulator， plus blocking IL-10 signaling， which can inhibit the IL-10 signaling in mice， did not increase the incidence of diabetes in DOB mice（P=0.6）.This result suggests that implying IL-10 inhibitor when inject vaccines may be safe. Maybe this is because IL-10 signaling is blocked for only a short period of time（26）.

My proposal

A 2017 study along with numerous of studies in 2010-2015 have showed that IL-10， especially CD4+T cells and CD8+T cells derived IL-10， can suppress the immune response to Mtb infections（14，19-21，25）. In addition， monocyte-derived IL-10， which is the most important source of IL-10 in Mtb infection， did not show significant effect in suppressing the immune response to Mtb infections（25）. So， we can try to preserve monocyte-derived IL-10 production after Mtb infection to reduce the potentially side effects.

In addition， a 2017 study showed that the inhibition of IL-10 for a short period of time in treating tumors may not cause serious side effects（23）. However， inhibiting or even reducing IL-10 signaling for a long period of time is very likely to cause serious side effects such as enteritis and rheumatic arthritis. So， I think a study to determine whether inhibit IL-10 signaling for even a short period of time， such as when injecting therapeutic vaccines or using anti-tuberculosis， will cause side effects in Mtb infections is urgent needed. If yes， then experiments that can determine the minimum concentration of IL-10 in human（maybe can use non-human primates model to determine） that is safe and also， at the same time， maximize the enhancement effect to T cells against TB infection is needed. If no， then the determination of the longest safe blockage time is needed.

Moreover， once determined， the safe range will be an important reference factor in future IL-10 inhibitor development for TB infections. Nonetheless， whether IL-10 inhibitor developed for TB will lead to serious side effect when applying to other diseases is remain unclear and therefore need further study. In conclusion， either possible situations need study immediately. Therefore， I proposed that the establishment of a laboratory team studying in this theme is important and needs quick action.

Also， the intake method may affect the efficacy of many tuberculosis drugs and vaccines， therefore very likely to including the potential IL-10 inhibitor. If the drugs are taken by oral or intramuscular， the effective factors need to travel longer to reach the place of Mtb infection though blood vessels indirectly. In contrast， an inhaled IL-10 inhibitor can reach the infected place quickly and directly， which may result in reaching the effective concentration quicker and better efficacy.

Finally， although we cannot just simply assumes that the inhibit of IL-10 can work for all diseases， however， IL-10 shows immunosuppression effects not only in Mtb infection， but also in a lot of other diseases such as toxoplasmosis （22）， leishmaniasis （23）， and malaria（24）.Therefore， I suggest that maybe we should conduct efficacy-determine tests to see if the inhibition of IL-10 when the vaccine inject or drugs taken can enhance vaccines' efficacy.

Reference

[1] WHO tuberculosis fact sheet detail page.http：//www.who.int/news-room/fact-sheets/detail/tuberculosis

[2] Lixia Wang， Hui Zhang， YunzhouRuan， Daniel P Chin， Yinyin Xia et.al.（June 2014） "Tuberculosis prevalence in China， 1990–2010；a longitudinal analysis of national survey data". Lancet383：2057-2064. doi：10.1016/S0140-6736（13）62639-2

[3] Cole ST， Brosch R， Parkhill J， Garnier T， Churcher C， Harris D， Gordon SV， et.al. （June 1998）. "Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence". Nature. 393 （6685）： 537–44. doi：10.1038/31159. PMID 9634230.

[4] Colditz GA， Berkey CS， Mosteller F， Brewer TF， Wilson ME， Burdick E，F(xiàn)ineberg HV（1995）： The efficacy of bacillus Calmette-Guérin vaccination ofnewborns and infants in the prevention of tuberculosis： meta-analysesof the published literature. Pediatrics， 96：29-35.

[5] Behr MA（2002）： BCG–different strains， different vaccines？ Lancet Infectious Disease2：86-92.

[6] Joel D. Ernst.（11.07.2018）： "Mechanisms of M. tuberculosis Immune Evasionas Challenges to TB Vaccine Design". Cell Host & Microbe 24：34-42.

[7] Eskdale J， Kube D， Tesch H， Gallagher G （1997）. "Mapping of the human IL10 gene and further characterization of the 5'flankingsequence". Immunogenetics. 46 （2）：120–8.doi：10.1007 /s002510050250.PMID 9162098.

[8] Zheng LM， Ojcius DM， Garaud F， Roth C， Maxwell E， Li Z， Rong H， Chen J， Wang XY， Catino JJ， King I （August 1996）. "Interleukin-10 inhibits tumor metastasis through an NK cell-dependent mechanism". The Journal of Experimental Medicine. 184 （2）：579–84. doi：10.1084/jem.184.2.579. PMC 2192723.PMID 8760811

[9] Sikka G， Miller KL， Steppan J， Pandey D， Jung SM， Fraser CD， Ellis C， Ross D， Vandegaer K， Bedja D， Gabrielson K， Walston JD， Berkowitz DE， Barouch LA （February 2013）. "Interleukin 10 knockout frail mice develop cardiac and vascular dysfunction with increased age". Experimental Gerontology. 48 （2）： 128–35. doi：10.1016/j.exger.2012.11.001.PMC 3744178， PMID 23159957

[10] Ozenci V， Kouwenhoven M， Huang YM， Xiao B， Kiviskk P， Fredrikson S， Link H （May 1999）. "Multiple sclerosis： levels of interleukin-10-secreting blood mononuclear cells are low in untreated patients but augmented during interferon-beta-1b treatment". Scandinavian Journal of Immunology. 49 （5）： 554–561. doi：10.1046/j.1365-3083.1999.00546.x.PMID 10320650

[11] Braat H， Rottiers P， Hommes DW， Huyghebaert N， Remaut E， Remon JP， van Deventer SJ， Neirynck S， Peppelenbosch MP， Steidler L （June 2006）. "A phase I trial with transgenic bacteria expressing interleukin-10 in Crohn's disease". Clinical Gastroenterology andHepatology. 4 （6）： 754–9. doi：10.1016/j.cgh.2006.03.028. PMID 16716759.

[12] Schreiber S， Fedorak RN， Nielsen OH， Wild G， Williams CN， Nikolaus S， Jacyna M， Lashner BA， Gangl A， Rutgeerts P， Isaacs K， van Deventer SJ， Koningsberger JC， Cohard M， LeBeaut A， Hanauer SB （December 2000）. "Safety and efficacy of recombinant human interleukin 10 in chronic active Crohn's disease. Crohn's Disease IL-10 Cooperative Study Group". Gastroenterology. 119 （6）：1461–72. doi：10.1053/gast.2000.20196.PMID 11113067.

[13] Infante JR， Naing A， Papadopoulos KP， Autio KA， Ott PA， Wong DJ， Falchook GS， Patel MR， Pant S （2015-05-20）. "A first-in-human dose escalation study of PEGylated recombinant human IL-10 （AM0010） in advanced solid tumors". ASCO Meeting Abstracts. 33 （15_suppl）： 3017.

[14] Redford， P. S.， P. J. Murray， and A. OGarra.（ 2011）. "The role of IL-10 in immuneregulation during M. tuberculosis infection". Mucosal Immunology4： 261–270.

[15] Verbon， A.， N. Juffermans， S. J. Van Deventer， P. Speelman， H. Van Deutekom，and T. Van Der Poll. 1999. Serum concentrations of cytokines in patients withactive tuberculosis （TB） and after treatment. Clin. Exp. Immunol. 115： 110–113.

[16] Bonecini-Almeida， M. G.， J. L. Ho， N. Boe′chat， R. C. Huard， S. Chitale， H. Doo，J. Geng， L. Rego， L. C. Lazzarini， A. L. Kritski， et al. 2004. Down-modulation oflung immune responses by interleukin-10 and transforming growth factor b（TGF-b） and analysis of TGF-b receptors I and II in active tuberculosis. Infect.Immun. 72： 2628–2634.

[17] Almeida， A. S.， P. M. Lago， N. Boechat， R. C. Huard， L. C. Lazzarini，A. R. Santos， M. Nociari， H. Zhu， B. M. Perez-Sweeney， H. Bang， et al. 2009. "Tuberculosis is associated with a down-modulatory lung immune response thatimpairs Th1-type immunity." Journal Immunology. 183： 718–731.

[18] Awomoyi， A. A.， A. Marchant， J. M. Howson， K. P. McAdam， J. M. Blackwell，and M. J. Newport. 2002. Interleukin-10， polymorphism in SLC11A1 （formerlyNRAMP1）， and susceptibility to tuberculosis. J. Infect. Dis. 186： 1808–1814.

[19] Zhang， M.， J. Gong， D. V. Iyer， B. E. Jones， R. L. Modlin， and P. F. Barnes. 1994.T cell cytokine responses in persons with tuberculosis and human immunodeficiency virus infection. J. Clin. Invest. 94： 2435–2442.

[20] OLeary， S.， M. P. OSullivan， and J. Keane. 2011. IL-10 blocks phagosomematuration in mycobacterium tuberculosis-infected human macrophages. Am. J.Respir. Cell Mol. Biol. 45： 172–180.

[21] Jonathan M. Pitt， Evangelos Stavropoulos， Paul S. Redford， Amy M. Beebe， Gregory J. Bancroft， Douglas B. Young and Anne OGarra "Blockade of IL-10 Signaling during Bacillus Calmette-Guérin Vaccination Enhances and Sustains Th1， Th17， and Innate Lymphoid IFN-γ and IL-17 Responses and Increases Protection toMycobacterium tuberculosis Infection"（October 15， 2012）Journal of Immunology， 189 （8） 4079-4087；DOI：https：//doi.org/10.4049/Journal immunology1201061