Table 4.

Human studies on ENL investigating cytokines.

Reference; study site(s)	Study population	Timing of sampling	MDT status	ENL treatment status	Type of samples	Measures	Findings
Filley et al. (168); India ε	7 ENL	Before, during and after the episode	All patients on MDT	ENL treated with steroids and/or thalidomide	Serum	IL2R	IL2R increase during ENL
Rea and Modlin (102); USA γ	ND	ND	ND	ND	Skin	IL-2 positive and IFN-γ positive mRNA-bearing lymphocytes	IL2- positive lymphocytes prevalent in ENL and in tuberculoid lesions
							Cells expressing IFN-γ mRNA in ENL lesions slightly increased compared to lepromatous
Sarno et al. (138); Brazil	18 ENL, 39 BT/BL/BB/LL, 4 T1R	ND	16/18 patients on various stages of MDT/2 untreated	3 ENL on thalidomide and 7 ENL on prednisone; others untreated for reaction	Serum	Tumor necrosis factor (TNF)-α and IL-1	TNF varied from undetectable to extremely high levels in ENL
							No correlation between severity of ENL and cytokine level
							Neither TNF nor IL-1 correlate with number or duration of ENL episodes
							Treated patients with steroids or thalidomide lower TNF
Sehgal et al. (172); India	11 ENL, 14 T1R, 20 leprosy non-reactional, 10 HC	Before starting antireactional treatment and when clinical signs of reaction had abated	On MDT	Samples before and after starting antireactional treatment	Serum	IL-2R	T1R upgrading group higher IL-2R than ENL
Sullivan et al. (173); USA ε	ND	ND	ND	ND	Skin	IFN-γ and TNF-α mRNA	IFN-γ mRNA in ENL similar to tuberculoid
							In LL and ENL lesions about 0.2% of cells expressed TNF-α
Barnes et al. (155); USA	12 active ENL, 14 inactive ENL, 6 T1R; 11 LL	ND	All patients had received less than 5 years chemotherapy	ND	PBMC	TNF-α	ENL: the levels of TNF-α release by PBMC were higher than any other leprosy
							Thalidomide reduced TNF-α by more than 90%
Parida et al. (140); India	12 ENL, 64 leprosy TT/BT/BB/BL/LL, 14 T1R	ND	Most patients before MDT treatment	ND	Serum	TNF and IL-1	Patients undergoing T1R or ENL showed high TNF levels
							Significant correlation between TNF and IL-1 in reaction
Sampaio et al. (150); Brazil and USA	13 LL ENL, 15 LL, 9 HC	ND	All patients were receiving MDT during the study.	7 ENL patient blood samples before starting treatment with thalidomide and 6 1-2 weeks after thalidomide	Plasma	TNF-α	ENL patients greater release of TNF-α from monocytes
					PBMC		High plasma TNF-α in ENL
					Monocytes
Bhattacharaya et al. (146); India	11 ENL, 14 T1R, 20 leprosy without reactions, 20 HC	Before treatment and after clinical remission of reaction	on MDT	Before antireactional treatment with steroids	Serum	TNF	TNF levels in acute ENL were higher but not significant and rose to become significant following treatment and clinical remission than HC and MB controls
Foss et al. (128); Brazil γ	28 lepromatous: 11 ENL, 23 tuberculoid, 19 HC	ND	86% of lepromatous patients treated with dapsone	Time of blood collection no immunosuppressive drug	Serum	TNF-α	TNF was elevated in the serum of ENL patients
Sampaio et al. (139); Brazil ε	49 BL/LL: 24 developed ENL	At the time of developing ENL, during thalidomide treatment, or after thalidomide treatment was discontinued; collected at 1-3, 6-7, and/or 13-21 days of thalidomide and 1-2 months after thalidomide	MDT was continued through the study	Thalidomide treatment for ENL	Sera	TNF-α, IL-6, IFN-γ	ENL highest TNF-α levels, which decreased significantly during thalidomide treatment Serum IFN-γ elevated in patients with high TNF-α levels
Santos et al. (156); Brazil	14 ENL (4 BL/10 LL), 12 BL/LL, 11 HC, 4 ENL post-reactions	ND	Half untreated and the other half treated with MDT	ENL patients were treated with thalidomide?	PBMC	TNF-α: spontaneous and M. leprae stimulated	ENL patients showed significantly greater release of TNF-α both spontaneously and induced by M. leprae-induced release in ENL patients
Vieira et al. (131); Brazil γ, ε	95 MB (30 LL/65 BL) of which 51 ENL	At leprosy diagnosis and at onset of reactional episode	Time of MDT for each ENL	Sample before thalidomide and steroids?	Serum	TNF-α, soluble IL-2R	TNF-α increased in 70.6% of ENL patients
Memon et al. (141); Pakistan	12 ENL, 27 leprosy (TT/BT/BL/LL), 14 household contacts and 22 endemic HC with no known leprosy contact	At the onset of ENL before initiation of treatment for reaction and after the reaction had subsided	10/12 ENL received previous MDT	Samples before antireactional treatment	Serum	TNF-α	TNF levels higher during acute phase of ENL and declined after clinical remission of the reaction
Moubasher et al. (148); Egypt	35 reactional (19 ENL/16 T1R), 55 leprosy, 20 HC	ND	Untreated ENL?	Untreated ENL?	Serum	IFN-γ, IL-2, IL-2R, IL-10, TNF-α, IL-1β	Both T1R and ENL showed significantly higher serum IFN-γ, IL-2R and IL-1β compared to non-reactional leprosy ENL showed increased levels of IL-10 compared to T1R
Moubasher et al. (165); Egypt	35 reactional (19 ENL), 36 non-reactional, 20 HC	PB patients assessed after 6 and 12 months of MDT/MB assessed after 12 months of MDT; Before and at the end of treatment with MDT	Before and after treatment with MDT	Corticosteroids were given to control the reactions	Serum	IL-2R, IL-10, IL-1β	IL-1β levels may have a prognostic marker for the development of reactions
Partida-Sanchez et al. (142); Mexico ε	9 ENL, 10 non-ENL, 10 HC	Beginning of reaction and after 1 and 2 months of thalidomide	All patients on MDT	Untreated samples and after 1 and 2 months of thalidomide	Serum	TNF-α, IFN-γ	TNF-α was significantly higher in ENL compared to non-ENL
							TNF levels decreased after ENL treatment
							IFN-γ significantly lower in patients at the onset of ENL and increased after thalidomide
Sampaio et al. (147); Brazil	18 MB with ENL (5BL/13LL)	Biopsies at diagnosis, at onset of reaction, and after 3 and/or 7 days of pentoxifylline; Serum: day 0 (during ENL), 3-7, 10-14, 30 and 60 days after pentoxifylline	7 patients with ENL newly diagnosed; others on MDT	Pentoxyfylline, 2 ENL patients on thalidomide	PBMC	Serum TNF-α, IL-6, IL-10	Elevated TNF-α in the sera of ENL
					Serum	TNF-α, IL-6, IL-10 release by PBMC following M. leprae stimulation or LPS stimulation	Treatment with pentoxifylline reduced TNF-α
							Serum levels of IL-6 increased during ENL
							High TNF-α mRNA expression in lesions during ENL which decreased following treatment with pentoxifylline
					Skin	TNF-α, IL-6, IL-10 gene expression at skin	IL-6 mRNA reduced by up to 50-fold after treatment
Moraes et al. (161); Brazil	53 leprosy: 20 ENL, 11 T1R	At the time of leprosy diagnosis (unreactional) and at the onset of first reactional episode (reactional)	MDT was continued through the study	No anti-inflammatory drugs at the time of sample collection	PBMC	IL-1β, IL-6, IL-8, GM-CSF, IFN-γ, IL-2Rp55, perforin, TNFβ, TNF-α mRNA in PBMC; IL-4, IL-6, IL-8, IL10, IL-12, IFNγ, TNFα mRNA in skin	In 7 ENL higher incidence of IFN-γ, perforin, GM-CSF, IL2R mRNA in blood
							Upregulation of IL-1β, IL-6, GM-CSF, IL-2R, IFN-γ mRNA in blood at onset of ENL at 3 ENL follow-up
				3 patients sequential sampling and after thalidomide	Skin		Skin lesions ENL: IFNγ and IL-4 differentially expressed
Oliveira et al. (33);Brazil α	10 BL/LL: 6 ENL, 10 HC	ND	On MDT	ND	Blood, P.B.Neutrophils	TNF-α, IL-8	Stimulated neutrophils secrete IL-8 and TNFα
							Increased TNF-α secretion from neutrophils after LPS stimulation
							Thalidomide inhibited TNF-α by neutrophils
Goulart et al. (174); Brazil	19 leprosy: 5 ENL/3 T1R, 9 HC	Untreated samples	Untreated samples	Untreated samples	PBMC	TGF-β1 in supernatants from adherent PBMC after stimulation with PGL-1, LPS or serum-free RPMI	Adherent PBMC from ENL secrete higher TGF-β1
Moraes et al. (164); Brazil	13 MB: 10 ENL, 3 T1R	Before and during pentoxyfilline or thalidomide	All patients on MDT	Before and during pentoxyfilline or thalidomide	Skin	mRNA expression: IFN-γ, IL-6, IL-10, IL-12 p40, TNF-α, IL-4	Expression of IFN-γ, IL-6, IL-10, IL-12 p40, TNF-α at the onset of reactional episodes (T1R and ENL) but IL-4 rarely detected Follow-up: TNF-α mRNA and IFN-γ, IL-6 and IL12p40 mRNA decreased after thalidomide or pentoxyfylline
Nath et al. (162); India	36 ENL, 105 TT/BL/LL 7T1R, 9 HC	ND	All patients on MDT	ENL patients before antireactional treatment	PMBC	IFN-γ, IL-4, IL-10, IL-12	ENL: 58% demonstrated a polarized Th1 pattern with only 30% expressing both cytokines
Nath et al. (163); India	1 BL/7 LL ENL, 2 BL/6 LL8 stable	ND	Most patients on MDT	ENL patients prior to antireactional therapy	PBMC	Real-time PCR forIFN-γ, IL4, IL10, p40 IL12	IFN-γ detectable in all and IL12p40 in half of ENL IL12p40 mRNA higher in ENL compared to stable lepromatous
Sampaio et al. (157); Brazil	15 leprosy: 10 ENL	ND	On MDT	ND	PBMC, monocytes, monocytes/T-lymphocytes cocultures	TNF-α after stimulation with M. leprae	Isolated monocytes from ENL released significantly more TNF-α in response to M. leprae than monocytes from non-reactional
Tadesse et al. (132); Ethiopia γ	14 BT, 11 BT T1R, 8 ENL, 11 HC	ND	ND	All ENL treated with steroids	PBMC	TNF-α in culture supernatants	Thalidomide resulted in suppression of TNF-α production
Haslett et al. (115); Nepal	20 ENL, 20 LL with no history of ENL within the preceding 30 days	Blood samples: days 0, 3, 7, 14, and 28 of thalidomide; ELISPOT: days 0, 7, 21, and 28	All (except 1 patient) on MDT	Excluded patients who had received immunomodulating therapy within the preceding month	Plasma	Plasma levels of IFN-γ, TNF-α, soluble IL2R, IL-12, IL-12 p40 and IL-12 p70	Circulating TNF-α levels lower at ENL diagnosis than controls
		Flow cytometry: days 0, 7, and 21; qRT-PCR: PBMCs days 0, 7, 21			T-cells	ELISPOT for IFN-γ; Flow cytometry for cytokine production by T cells	Upward trend during thalidomide ENL baseline plasma levels of IL-12 lower than control
					Skin	qPCR: IL-2 genes	Baseline levels of sIL2R higher in ENL than controls
							Thalidomide increased T cell subsets expressing both IL-2 and IFN-γ
Villahermosa et al. (134); Philippines γ, ε	22 ENL	Before thalidomide and at study weeks 3 and 7 during thalidomide	MDT was continued	Samples untreated for antireactional drugs and during thalidomide	Plasma	TNF-α, IL-6	TNF-α levels not detected
							IL-6 unchanged or reduced following thalidomide from week 0 to week 3
							IL-6 undetectable at weeks 3 and 7
Belgaumkar et al. (169); India	71 BT/BB/BL, 11 pure neuritic, 6 T1R, 1 ENL, 30 HC	Untreated samples	Untreated samples	Patients on antileprosy treatment or steroids were excluded	Serum	IL-6, IFN-γ	The one patient with ENL had higher levels of IL-6 and IFN-γ in comparison to the BL/LL patients without reactions
Iyer et al. (143); Indonesia ε	131 TT/BT/BB/BL/LL, 44 ENL, 5 T1R, 112 HC	ND	Patients on MDT	Prednisolone to treat reactions	Serum	IL-6, IFN-γ, TNF-α, IL-6R, IL-10, IL-4, sCD27	IFN-γ and IL-6R increased in ENL compared to non-ENL
							Completion of corticosteroid treatment: IFN-γ, TNF-α, sIL6R declined
Stefani et al. (151); Brazil	10 ENL, 10 T1R, 29 non-reactional controls	Newly detected untreated patients	Untreated samples	Untreated samples	Plasma	TNF-α, IFN-γ, IL12p70, IL-2, IL-17, IL-1β, IL-6, IL-15, IL-5, IL-8, MIP-α, MIP-β, RANTES, MCPI, CCL11/eotaxin, CXCL10, IL-4, IL-10, IL13, IL-1Rα, IL-7, IL-9, G-CSF, PDGF BB, bFGF, VEGF	IL-6, IL-7 and PDGF BB elevated in ENL
Motta et al. (175); Brazil	44 leprosy of which 15 ENL, 10 HC	Baseline and 7 days after therapy for oral infection	ND	ND	Serum	IL-1, TNF-α, IL-6, IFN-γ, IL-10	No specific finding for ENL
Teles et al. (176); Brazil ε	32 leprosy: 10 ENL, 8 T1R	4 ENL patients before and during reaction	All patients on MDT	ND	Skin	TNF-α gene expression and levels in supernatants	PBMC stimulated with M. leprae: upregulation of gene expression of TNF-α and increase of TNF-α in supernatants after 1, 3, and 6 h
					PBMC
Jadhav et al. (149); India ε	303 MB: 5 ENL	Serum samples at the time of recruitment	Newly registered: no MDT	Untreated	Serum	TNF-α	No significant outcome for ENL
Madan et al. (144); India	61 leprosy: 4 ENL and 2 ENL during study	Untreated samples, during reactional episodes and after completion of treatment	Untreated patients	Patients on steroids were excluded	Serum	TNF-α, IFN-γ, IL-1β, IL-10	All cytokines were raised in reactional (both T1R and ENL) compared to non-reactional IFN-γ, IL-1β and IL-10 were higher in ENL but only IL-10 was statistically significant compared to T1R
							Levels of all cytokines decreased after MDT
Rodrigues et al. (145); Brazil	18 LL with ENL during treatment; 13 non-reactional BT, 37 non-reactional BL/LL, 25 BL with T1R during treatment; 21 HC	Beginning of leprosy treatment, at diagnosis of reactional episode and at 3-5 years post-treatment	Samples before and during MDT	Untreated samples and after treatment with prednisolone	Serum	TNF-α	TNF-α higher during ENL than prior to the reaction
Chaitanya et al. (177); India	21 ENL, 80 T1R, 80 leprosy without reaction, 94 non-leprosy	Untreated samples	Untreated samples	Untreated samples	Serum	IL-17F	IL-17F elevated during T1R but no significant difference in ENL
Lockwood et al. (178); India ε	303 MB leprosy: 13 ENL	Skin biopsies at enrollment	Before MDT	Before antireactional treatment	Skin	TNF-α and TGF-β immunostaining	TNF-α: similar levels ENL and non-ENL TGF-β: no difference in ENL and non-ENL
Martiniuk et al. (179); Nepal and USA ε	7 ENL	Pre- and post- treatment with thalidomide	ND	Pre- and post- treatment with thalidomide	Skin biopsies	RT-PCR for hIL-17A, hIL-17B, hIL-17C, hIL-17D, hIL-17E, hIL17F	IL17A, was consistently seen before and after thalidomide
							Reduction in IL17B, IL17E and increase of IL17C following thalidomide
Sousa et al. (170); Brazil	33 ENL, 54 T1R, 16 reaction-free leprosy	ND	63.8% presented ENL during MDT	ND	Plasma	IL-6	Higher IL-6 in ENL and T1R compared to non-reactional
Abdallah et al. (171); Egypt	43 leprosy: 6 ENL, 43 HC	Untreated samples	Untreated samples	Untreated samples	Serum	IL-17, IL-4	Overproduction of IL-4 in LL patients
Saini et al. (136); India γ	21 MB: 16 ENL, 5 T1R	ENL blood during reaction and at 0.5 and 1 year after the onset of reaction	Duration of MDT described	ENL patients received steroids	PBMC	PBMC stimulated with M. leprae, recombinant Lsr2 and 6 synthetic peptides spanning the Lsr2 sequence: IFN-γ	During ENL stimulated PBMC showed IFN-γ release
Abdallah et al. (109); Egypt γ	43 leprosy: 6 ENL, 40 HC	Untreated patients	Untreated samples	Untreated samples	Serum	IL-1β, IL-4, IL12p70, IFN-γ	IL-4 highest among LL compared to ENL
Attia et al. (110); Egypt γ	43 leprosy: 6 ENL, 40 HC	Untreated samples	Untreated samples	Untreated samples	Serum	IL-17, IL-22, IL-10, TGF-β	No statistically significant difference between groups
Berrington et al. (167); Nepal	85 leprosy: 9 ENL, 35 BL/LL non-reactional	ND	ND	ND	Skin	RT-PCR for CCL1, CCL2, CCL17, CCL18, IFNA1, IFNA8, IFNB1, IFNG, IL10, IL12a, IL12b, IL13, IL17a, IL18, IL1b, IL1ra, IL21, IL22, IL23, IL27, IL29, IL4, IL6, TNF	CCL18, IL12b and CD14 elevated in lesions of ENL but failed to reach significance when adjusted for multiple comparisons
Sallam et al. (166); Egypt	43 leprosy: 6 ENL, 43 HC	Untreated samples	Untreated samples	Untreated samples, excluded patients on corticosteroids	Serum	IL-1β, IL-12	Higher IL-1β in ENL compared to non- reactional
							No significant difference for IL-12
Dupnik et al. (58); Brazil β, ε	11 ENL, 11 T1R, 19 leprosy without reactions for microarray; 6 ENL, 11 T1R, 11 non-reactional for qPCR; 3 ENL for ICH	ND	3/11 ENL pre-treatment, 2/11 ENL on treatment and 6/11 post-treatment; leprosy controls matched for length of treatment	Excluded patients on steroids within 7 days and thalidomide within 28 days of enrollment	PBMC	Microarrays followed by qPCR	Cytokine-cytokine receptor interaction has been in the top 3 KEGG pathways in ENL CCL5 followed by IFN-γ was the most significant upstream regulator of the expression changes in the array
Saini et al. (113); India γ	66 leprosy: 15 T1R, 15 ENL, 36 stable leprosy without previous history or clinical evidence of reactions	Newly diagnosed leprosy patients prior to institution of antireaction therapy	Untreated samples	Untreated samples	PBMC	Antigen (MLSA) stimulated and unstimulated PBMC: gene expression with PCR array for 84 genes ELISA for cytokines IL-17A/F, IL-21, IL-22, IL-23A, IL-6, IL-1β, IFN-γ, TGF-β in supernatants	IL-23A mRNA expression increased in ENL IL-23R expression increased in ENL High expression of CCL20 and CCL22 in ENL ENL significant fold increase in IFN-γ Culture supernatants: Higher IL-17A/F in ENL patients compared to LL IL23A increased compared to LL IL-1β increased in ENL
Dias et al. (80); Brazil ε	30 ENL, 24 BL/LL, 31 HC	Upon diagnosis of reaction	BL/LL before MDT but most ENL patients on MDT	Before treatment with thalidomide or steroids	PBMC	TNF, IL-6 and IL-1β in response to TLR9 agonist	Higher production of TNF-α, IL-6, IL-1β in response to TLR9 agonist
							TLR9 antagonist inhibited the secretion of cytokines in response to M. leprae lysate

α, also in Table 1; β, also in Table 2; γ, also in Table 3; ε, also in Table 5.

BB, mid-borderline leprosy; BL, borderline lepromatous leprosy; BT, borderline tuberculoid leprosy; ENL, erythema nodosum leprosum; HC, healthy controls; ICs, immune complexes; LL, lepromatous leprosy polar; ND, not described; P.B.neutrophils, peripheral blood neutrophils; SLE, systemic lupus erythematosus; TB, tuberculosis; TT, tuberculoid leprosy polar.