Differential Expression of Circulating MiRNAs After Alemtuzumab Induction Therapy in Lung Transplantation

doi:10.21203/rs.3.rs-961660/v1

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Differential Expression of Circulating MiRNAs After Alemtuzumab Induction Therapy in Lung Transplantation

https://doi.org/10.21203/rs.3.rs-961660/v1

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Alemtuzumab is a monoclonal antibody targeting CD52, used as induction therapy after lung transplantation (LTx). Its engagement produces a long-lasting immunodepletion; however, the mechanisms driving cell reconstitution are poorly defined. We hypothesized that miRNAs are involved in this process.

The expression of a set of miRNAs, cytokines and co-signaling molecules was measured with RT-qPCR and flow cytometry in prospectively collected serum samples of LTx recipients, after alemtuzumab or no induction therapy.

Twenty-six LTx recipients who received alemtuzumab and twenty-seven matched LTx recipients without induction therapy were included in the analysis. One year after transplantation four miRNAs were differentially regulated: miR-23b (p=0.05) miR-146 (p=0.04), miR-155 (p<0.001) and miR-486 (p<0.001). Expression of 3 miRNAs changed within the alemtuzumab group: miR-146 (p<0.001), miR-155 (p<0.001) and miR-31 (p<0.001). Levels of IL-13, IL-4, IFN-γ, BAFF, IL-5, IL-9, IL-17F, IL-17A and IL-22 were different one year after transplantation compared to baseline. In no-induction group, concentration of sCD27, sB7.2 and sPD-L1 increased overtime.

Expression of miR-23b, miR-146, miR-486, miR-155 and miR-31 was different in LTx recipients who received alemtuzumab compared to recipients without induction therapy. The observed cytokine pattern suggested proliferation of specific B cell subsets in alemtuzumab group and co-stimulation of T-cells in no-induction group.

Molecular Genetics

Internal Medicine

alemtuzumab

(LTx)

RT-qPCR

LTxrecipients

miR-23b

miR-146

miR-486

miR-155

miR-31

Current immunosuppression (IS) protocols in lung transplantation (LTx) usually consists of an induction therapy and a triple-drug maintenance immunosuppression including a calcineurin-inhibitor, an antiproliferative drug and steroids¹. Currently, IL-2 receptor antagonists are used by the majority of centers as induction agents¹, followed by anti-thymocyte globulin (ATG) and alemtuzumab. Alemtuzumab is a humanized monoclonal antibody targeting CD52, a surface receptor expressed on CD4⁺ and CD8⁺ lymphocytes, Natural Killer (NK) cells and monocytes. Its activation induces cell lysis leading to an immune cell depletion lasting 6 to 24 months^2,3. Although composition of T and B cell populations after alemtuzumab induction in transplantation has been described in a few studies, the underlying mechanisms driving cell reconstitution are still unknown. Alemtuzumab was introduced in our clinical routine more than 10 years ago following a prospective randomized controlled trial comparing alemtuzumab and ATG⁴. This study showed a reduced incidence of higher grade ACR in the alemtuzumab group. Similarly, other groups observed superiority of alemtuzumab in terms of graft and patient survival^5,6. The mechanisms underlying the effects of alemtuzumab in the early induction phase as well as in the later immune reconstitution phase have not been clarified yet and a better knowledge of the key molecular players could open up further therapeutic possibilities.

MicroRNAs (miRNAs) are evolutionarily conserved small non-coding RNAs, which function as post-transcriptional negative regulators of target mRNAs⁷. As such, they play a fundamental role as orchestrators of complex biological processes including cell differentiation and apoptosis⁷. Aberrant expression of miRNAs is associated with initiation and progression of pathological processes including immune-mediated disorders, cancer and fibrosis^8–10. Moreover, miRNAs have been found to be involved in regulation of differentiation and proliferation of specific immune subsets.

The aim of the current study was to investigate the expression of a panel of miRNAs and cytokines in lung transplant recipients who received alemtuzumab induction therapy. Our hypothesis is that microRNA profiles in the two population of subjects might differ substantially, thus suggesting possible mechanistic effects of alemtuzumab induction therapy.

Demographics

Sixty patients (30 vs 30) were included in the analysis. Seven patients (4 in the control group and 3 in the alemtuzumab group) had to be excluded, since at least one microRNA was not detectable at one time point. Baseline characteristics were similar among the groups (Table 1). Median age was 58 years (IQR: 55-60) in the control group compared to 52 years (IQR: 46-59) in the alemtuzumab group. The most represented underlying diagnosis was chronic obstructive lung disease in both groups. Approximately 20% of recipients in both groups had a high risk CMV match (D-/R+). Table 1 summarizes patient characteristics of both study groups.

Table 1

– Group demographics
Parameters		Groups
Parameters		Controls (n=26)	Alemtuzumab (n=27)	p-value
Female (n, %)		14, 54%	15, 56%	0.692
Age (median, IQR)		58 (55-60)	52 (46-59)	0.520
Underlying diagnosis (n, %)	Obstructive diseases	22, 85%	16, 59%	0.163
	Fibrosis	2, 7%	5, 19%
	PH	1, 4%	2, 7%
	CF	0	3, 11%
	Others	1, 4%	1, 4%
CMV risk (n, %)	D+/R-	5, 19%	6, 22%	0.400
	D+/R+	9, 35%	12, 44%
	D-/R+	8, 31%	4, 15%
	D-/R-	4, 15%	5, 19%
Bilateral lung transplantation (n, %)		26, 100%	27, 100%	-
PGD at 72 hours (n, %)	0	20, 77%	19 (70%)	0.879
	1	4, 15%	4 (15%)
	2	1, 4%	2 (7%)
	3	0	0
	Ungradable	1, 4%	2 (7%)
High grade ACR (n, %)		7, 29%	0	0.003
High grade LB (n, %)		10, 42%	4, 15%	0.032
Cumulative A score (n, %)		0.11 (0-0.24)	0 (0-0.007)	0.002
Cumulative B score (n, %)		0.63 (0.39-0.92)	0.31 (0-14-0.59)	0.003

Longitudinal miRNAs expression

The no-induction group did not show any change in expression of miRNAs overtime. Instead, patients who received alemtuzumab had significantly higher expression of miR-146a and miR-155 one year after transplantation (1.5-fold change, p<0.001 and p<0.001, respectively) (Table 2, Figure 1). MiR-31 was found downregulated one year after transplantation in the alemtuzumab group (8-fold change, p<0.001, Figure 2).

Table 2

– MicroRNA expression differences within the groups between the baseline and one after transplantation
Timepoint	miRNA	Groups
		Baseline			One year after transplantation			p-value
		Median	Percentile 25	Percentile 75	Median	Percentile 25	Percentile 75	p-value
No-induction	miR-30b	0.636	0.271	1.082	0.433	0.011	1.329	0.578
	miR-24	0.034	0.007	0.047	0.026	0.002	0.055	0.821
	miR-98	0.892	0.243	1.117	0.639	0.047	1.161	0.226
	miR-99a	6.664	2.874	12.910	5.819	0.310	13.320	0.680
	miR-23b	6.002	1.749	8.048	4.085	0.241	10.478	0.606
	miR-125a	0.309	0.013	0.520	0.178	0.016	0.456	1.000
	miR-182	0.009	0.002	0.011	0.005	0.002	0.015	0.342
	miR-17	0.039	0.013	0.044	0.018	0.001	0.053	0.216
	miR-486	0.190	0.138	0.228	0.089	0.015	0.287	0.312
	miR-21	0.590	0.144	1.712	0.463	0.027	1.055	0.578
	miR-146a	0.067	0.020	0.108	0.042	0.006	0.135	0.381
	miR-155	0.006	0.002	0.015	0.005	0.000	0.022	0.765
	miR-31	6.275	3.903	17.957	4.221	0.621	13.619	0.337
Alemtuzumab	miR-30b	0.171	0.007	0.562	0.202	0.069	1.168	0.684
	miR-24	0.010	0.001	0.028	0.009	0.003	0.039	0.802
	miR-98	0.445	0.023	0.990	0.557	0.151	1.641	0.522
	miR-99a	13.985	0.828	46.339	9.165	3.510	26.038	0.831
	miR-23b	6.930	0.633	26.653	8.007	2.694	40.027	0.373
	miR-125a	0.036	0.008	0.165	0.091	0.020	0.154	0.802
	miR-182	0.001	0.000	0.007	0.003	0.001	0.017	0.377
	miR-17	0.008	0.000	0.040	0.011	0.003	0.029	0.938
	miR-486	0.025	0.007	0.088	0.030	0.006	0.087	0.831
	miR-21	0.128	0.004	0.244	0.116	0.004	0.481	0.654
	miR-146a	0.027	0.001	0.124	1.425	0.662	1.990	0.000
	miR-155	0.002	0.000	0.032	1.532	0.382	3.250	0.000
	miR-31	8.190	4.428	10.373	0.898	0.580	2.305	0.000

Comparison of miRNA expression between groups

One year after transplantation, four miRNAs showed different expression levels among the groups: miR-23b, miR-486, miR-146a and miR-155 (Figure 2, Table 3). MiR-23b was upregulated with a 4-fold change in the control group and an 8-fold change in the alemtuzumab group (p=0.046). Both miR-146 and miR-155 were upregulated in alemtuzumab groups and downregulated in the no-induction group (p<0.001 and p<0.001, respectively). MiR-486 was downregulated in both groups but the fold-change difference was negligible.

Table 3

– MicroRNA expression differences between the groups at baseline and one year after transplantation
Timepoint	miRNA	Groups
		No-induction			Alemtuzumab			p-value
		Median	Percentile 25	Percentile 75	Median	Percentile 25	Percentile 75	p-value
Baseline	miR-30b	0.636	0.271	1.082	0.171	0.007	0.562	0.097
	miR-24	0.034	0.007	0.047	0.010	0.001	0.028	0.215
	miR-98	0.892	0.243	1.117	0.445	0.023	0.990	0.288
	miR-99a	6.664	2.874	12.910	13.985	0.828	46.339	0.728
	miR-23b	6.002	1.749	8.048	6.930	0.633	26.653	0.636
	miR-125a	0.309	0.013	0.520	0.036	0.008	0.165	0.157
	miR-182	0.009	0.002	0.011	0.001	0.000	0.007	0.076
	miR-17	0.039	0.013	0.044	0.008	0.000	0.040	0.157
	miR-486	0.190	0.138	0.228	0.025	0.007	0.088	0.016
	miR-21	0.590	0.144	1.712	0.128	0.004	0.244	0.110
	miR-146a	0.067	0.020	0.108	0.027	0.001	0.124	0.475
	miR-155	0.006	0.002	0.015	0.002	0.000	0.032	0.875
	miR-31	6.275	3.903	17.957	8.190	4.428	10.373	0.928
One year after transplantation	miR-30b	0.433	0.011	1.329	0.202	0.069	1.168	0.450
	miR-24	0.026	0.002	0.055	0.009	0.003	0.039	0.234
	miR-98	0.639	0.047	1.161	0.557	0.151	1.641	0.831
	miR-99a	5.819	0.310	13.320	9.165	3.510	26.038	0.161
	miR-23b	4.085	0.241	10.478	8.007	2.694	40.027	0.046
	miR-125a	0.178	0.016	0.456	0.091	0.020	0.154	0.122
	miR-182	0.005	0.002	0.015	0.003	0.001	0.017	0.629
	miR-17	0.018	0.001	0.053	0.011	0.003	0.029	0.299
	miR-486	0.089	0.015	0.287	0.030	0.006	0.087	0.036
	miR-21	0.463	0.027	1.055	0.116	0.004	0.481	0.120
	miR-146a	0.042	0.006	0.135	1.425	0.662	1.990	0.000
	miR-155	0.005	0.000	0.022	1.532	0.382	3.250	0.000
	miR-31	4.221	0.621	13.619	0.898	0.580	2.305	0.096

Cytokine levels

In no-induction group, IL-13 (39 pg/ml, IQR 31-48 vs 0.1 pg/ml, IQR 0.1-28, p=0.002), IL-4 (18 pg/ml, IQR 13-28 vs 0.1 pg/ml, IQR 9-53, p<0.001), IL-5 (46 pg/ml, IQR 25-72 vs 24 pg/ml, IQR 0.1-54, p=0.031), IL-9 (22 pg/ml, IQR 17-29 vs 9 pg/ml, IQR 0.1-23, p=0.006), IL-17F (27 pg/ml, IQR 12-36 vs 0.1 pg/ml, IQR 0.1-21, p=0.001) and IL-22 (44 pg/ml, IQR 31-61 vs 30 pg/ml, IQR 0.1-54, p=0.033) showed lower levels one year after transplantation compared to baseline (Figure 3, Table S1). On the contrary, the concentration of IFN-γ (65 pg/ml, IQR 40-102 vs 183 pg/ml, IQR 62-440, p=0.003) and BAFF (0.1 pg/ml, IQR 0.1-628 vs 998 pg/ml, IQR 0.1-9586, p=0.008) increased overtime. Similarly, in this group, sCD27 (38,161 pg/ml, IQR 29,532-70,727 vs 91,385 pg/ml, IQR 67,772-139,278, p=0.026), B7.2 (66 pg/ml, IQR 59-96 vs 152 pg/ml, IQR 115-192, p=0.001) and sPD-L1 (38 pg/ml, IQR 31-44 vs 48 pg/ml, IQR 37-55, p=0.042) showed higher concentration levels at one-year post-transplant (Table S2).

Patients who received alemtuzumab had lower serum levels of IL-13 (34 pg/ml, IQR 9-43 vs 0.1 pg/ml, IQR 0.1-29, p=0.041), IL-4 (12 pg/ml, IQR 0.1-20 vs 0.1 pg/ml, IQR 0.1-0.1, p=0.005), IL-17A (4 pg/ml, IQR 0.1-8 vs 0.1 pg/ml, IQR 0.1-4, p=0.043), IL-9 (17 pg/ml, IQR 0.1-32 vs 0.1 pg/ml, IQR 0.1-17, p=0.026) and IL-22 (23 pg/ml, IQR 4-38 vs 0.1 pg/ml, IQR 0.1-17, p=0.015) one year after transplantation (Figure 4) with respect to baseline. Instead, levels of BAFF (3934 pg/ml, IQR 0.1-9270 vs 10232 pg/ml, IQR 4436-17518, p=0.015) and IFN-γ (23 pg/ml, IQR 4-38 vs 0.1 pg/ml, IQR 0.1-17, p=0.015) increased overtime (Table S1).

Correlations

One year after transplantation, BAFF positively correlated with miR-146a (r=0.288, p=0.040) and miR-155 (r=0.261, p=0.050) while IL-17A negatively correlated with miR-155 (r=0.418, p=0.033) in alemtuzumab group. No other significant correlations were observed.

MiRNA gene target prediction and pathway enrichment analysis

After identification of differentially regulated miRNAs, gene target prediction analysis was performed. The total number of identified gene targets after accounting for overlap was 10,461. Using the identified gene targets, we performed enrichment analysis to identify significantly enriched pathways using the KEGG database. MultiMir revealed 39 significantly enriched pathways with a p-value < 0.05 and a false discovery rate (FDR) < 0.10 (Benjamini-Hochberg procedure) in alemtuzumab group compared to controls at one year after transplantation. Similarly, 20 significantly enriched pathways were identified at one year after transplantation compared to baseline in alemtuzumab patients. The pathway names, odds ratios and levels of significance are listed in Table S3 and S4. Analysis of the identified gene targets showed that signaling pathways involved in immune cell proliferation were enriched: TLRs signaling pathway, cytokine downstream signaling pathways, mTOR pathway, Jak-STAT signaling pathway, MAPK pathway and PI3K-Akt signaling pathway (Table S3 and S4). Moreover, transcription factors fundamental in immune cell activation such as forkhead box protein O1 (FOXO1), signal transducer and activator of transcription 1 and 5 (STAT1 and STAT 5), suppressor of cytokine signaling 1 (SOCS 1), nuclear factor kappa B (NF-kB), TNF receptor associated factor 6 (TRAF 6), interleukin-1 receptor-associated kinase 1 (IRAK 1), bHLH transcription factor (c-Myc) and SMAD family members were found as main targets of the differentially regulated miRNAs.

Over the last 20 years, constant refinements of the immunosuppression protocols contributed to the improvement of long-term outcome after lung transplantation. Currently, IS protocols include an induction therapy and a triple-drug maintenance immunosuppression, constituted by a calcineurin-inhibitor, an anti-proliferative agent and steroids¹. Alemtuzumab is a monoclonal antibody, recently used as induction agent in solid organ transplantation. Based on the published experience, this agent shows excellent results in terms of rejection rates and survival^4–6. MiRNAs are post-transcriptional regulators of complex biological and pathological processes and have been identified to exert a key function in hematopoiesis and immune cell homeostasis and activation. In the current study, we identified a set of differentially regulated miRNAs (miR-155, miR-146a, miR-23b, miR-31 and miR-486) one year after alemtuzumab induction therapy. Moreover, a panel of cytokines were found to be differently expressed one year after transplantation in both no-induction and alemtuzumab. Only in the alemtuzumab group, BAFF and IL-17A showed a positive and negative correlations with miR-146 and miR-155, respectively. Finally, enrichment pathway analysis supported the hypothesis that these five miRNAs are key regulators of immune cell proliferation, lineage commitment and inflammatory response.

Alemtuzumab is a monoclonal antibody against CD52, which is a glycoprotein found on the surface of lymphocytes, monocytes and dendritic cells. The engagement of alemtuzumab on CD52 causes a profound long-lasting immunodepletion ^2,3. B cell counts recover within 3 to 6 months and T cell counts within 12 to 24 months after treatment ^2,11. After reconstitution, effector memory T cells and CD25⁺FoxP3⁺ Tregs seem to constitute the predominant T cell subsets ^11–16. The B cell compartment mainly consists of IgM-producing näive B cells and transient B cells ^2,11. Such phenotypic changes play a crucial role in allograft tolerance mechanisms¹⁷.

MicroRNAs are known key players in the pathogenesis of autoimmune diseases, fibrosis and cancer but their role in the context of immunosuppression after lung transplantation has not been studied yet. For the first time, could show herein that miR-155 and miR-146a, two key orchestrators of hematopoiesis and immune cell function are upregulated one year after alemtuzumab treatment. MiR-155 controls T-cell lineage commitment, by promoting T helper type 1 (Th1) over T helper type 2 (Th2) cells, most likely by targeting the transcription factor c-Maf and negatively modulating the transcription of type 2 cytokines¹⁸. It has been previously described that deficiency of miR-155 leads to decreased germinal center (GC) size and response, lower levels of plasma and memory cells and impaired production of high-affinity class-switched IgGs^18–20. MiR-146a is dynamically regulated during CD4⁺ cell differentiation, with an upregulation in differentiated Th1 and a downregulation in Th2 cells²¹. Furthermore, miR-146a regulates survival and maturation of dendritic cells (DC)²². Thus, it is reasonable to hypothesize that both miRNAs may actively shape the reconstitution of immune cell subsets observed after alemtuzumab treatment^11,14−16.

B-cell activating factor is a cytokine belonging to TNF family, emerged as a critical factor for B cell survival and maturation²³. Three main receptors of BAFF are known: BCMA, TACI and BAFF-R. BAFF binds strongly to TACI and BAFF-R but only weakly with BCMA²⁴. BAFF-R is critical for survival and maturation of B cell precursors, it is expressed at late transitional stage and is present on all mature B cells. Its expression is decreased in B cells in the GC reaction but is expressed again on memory B cells. It is absent on plasmablasts and plasma cells²⁵. TACI is present on late transitional stage and BCMA is present on plasma cells and is responsible for their long-lived survival^26–28. The cytokine APRIL shares the receptor BCMA and TACI with BAFF, however, contrarily to BAFF, it strongly binds to BCMA and weakly to TACI. This slightly different expression pattern independently regulates homeostatic proliferation of different B-cell subsets²⁹. In our cohort, we observed a strong increase in BAFF expression but not of APRIL. Concomitantly, serum levels of sCD40L and IL-6, which are both required for B cell maturation in plasma cells and IL-6 production^30,31, did not increase overtime. Thus, the isolated increased expression of BAFF may indicate the proliferation of memory B cells, a cell subset which is spared by alemtuzumab^2,11. Similar to our findings, serum BAFF levels but not APRIL were found to be increased in a cohort of kidney recipients after alemtuzumab induction³². Interestingly, the authors observed that BAFF in combination with sirolimus was able to drastically increase activation of B-cells whereas in the presence of CNI, BAFF had only little effect on B-cell activation. We found a positive correlation between miR-155 and BAFF. A similar finding was observed in rheumatoid arthritis patients, whose CD19⁺ B cells expressed miR-155 under stimulation by BAFF³³. As miR-155 plays a critical role in early B cell commitment and proliferation, it is reasonable to hypothesize that, after alemtuzumab immunodepletion, the memory B cells compartment undergoes a homeostatic proliferation driven by miR-155 through the mediation of BAFF. However, this mechanism needs to be further clarified in future experiments.

In the current study, miR-31 was found to be significantly down-regulated in alemtuzumab patients. It is well described that miR-31 positively correlates with T cell activation and induces the transcription of IL-2 in T cells by suppression of RhoA and activation of nuclear factor of activated T-cells (NF-AT)^34,35. Moreover, miR-31 directly targets IL-25 transcription, with the consequent production of IL-12 and IL-23 by antigen-presenting cells and an increased Th1/Th17 response³⁶. Thus, downregulation of miR-31 may significantly contribute to contain T cell activation. An analogous effect can be ascribed to the upregulation of miR-146a, an anti-inflammatory microRNA, which we found in the alemtuzumab group. Engagement of T-cell receptor (TCR) triggers expression of miR-146a which negatively targets NF-kB and inflammatory response ³⁷. Knockout of miR-146 is associated to increased IFN-γ production and consequently Th1 cell response³⁸. Moreover, miR-146 has emerged as negative master regulator of downstream signaling of Toll-like receptors, tumor necrosis factor and IL-1 receptor by targeting TRAF6/IRAK1/NF-kB pathway. Taken together, it is reasonable to hypothesize that the downregulation of miR-31 and upregulation of miR-146 might participate in the regulation of T cell responses and the Tregs/Th17 balance, thus favoring graft tolerance. Alemtuzumab, in fact, has been associated with an increase in peripheral levels of anti-inflammatory cytokines and with a suppression of Th1 and Th17 transcription factors^12,13. This is further supported in our findings by the negative correlation found between miR-155 and IL-17A.

The frequency of Treg cells seems to increase after alemtuzumab immunodepletion¹⁴. A similar finding was also reported by Bloom et al. in kidney transplant recipients¹⁵. Furthermore, Noris and colleagues observed an increase of Tregs in kidney transplant recipient treated with alemtuzumab up to 24 months after depletion therapy¹¹. MicroRNAs have been found to be fundamental regulators of Tregs commitment and proliferation³⁹. MiR-155 binds to the promoter region of FoxP3⁴⁰. Increased FoxP3 transcription induces miR-155 expression, which further stimulates Tregs proliferation in a feedback loop⁴¹. Rudensky’s group also showed that miR-146 is upregulated in Tregs³⁸. MiR-31 is known to target FoxP3 mRNA and negatively influence its transcription⁴², on the contrary miR-23 is a FoxP3 transcriptional target and its expression is involved in Tregs commitment⁴³. This is in line with our observation of an upregulation of pro-tolerogenic miR-155/-146/-23b and downregulation of anti-tolerogenic miR-31 in the alemtuzumab group at 1 year after transplantation.

As ancillary finding, serum levels of three soluble co-signaling molecules, namely sCD27, B7.2 and sPD-L1, were increased one year after transplantation in no-induction group while remained unchanged in alemtuzumab group. Soluble CD27 is secreted by activated T-cells and plays a role in their maturation and proliferation. Increased levels have been detected in inflammatory and autoimmune diseases, viral infections and lymphoid malignancies, making it a potential marker for T-cell–mediated inflammation⁴⁴. Similarly, soluble B7.2 is a costimulatory molecule of human T cells⁴⁵ and it has been found increased in systemic lupus erythematosus, asthma and leukemia^46–48. It is reasonable to hypothesize that soluble B7.2 could have a similar stimulatory effect in no-induction group. Finally, higher levels of sPD-L1 were associated with poorer long-term graft outcome in kidney recipients⁴⁹. The soluble form of this molecule may play as antagonist of PD-1/PD-L1 engagement, thereby stimulating proliferation and cytokine production of T cells⁵⁰.

Our study is not free of limitations. First, quantification of immune cell subsets by FACS is lacking. This could have strengthened our results, by correlating the presented molecular signature with the cellular findings^2,11. Further, serum samples beyond one year after transplantation were not available for analysis. This would have deepened our knowledge about the repopulation of other immune cell subsets as well as the possible long-term effects of induction therapy. Furthermore, the explanatory and pilot nature of the study may solely suggest possible underlying processes, which have to be further confirmed by mechanistic studies. On the other side, it should be acknowledged that the current analysis represents the first study describing different miRNAs expression after LTx in a prospectively collected cohort after alemtuzumab induction.

In conclusion, this report provides the first evidence of miRNAs and cytokine dysregulation following alemtuzumab induction therapy in lung transplantation. Since these dysregulated mediators play a role in the homeostatic reconstitution of different B cell subsets and in the regulation of T cell response, their role in regulation of graft tolerance and in humoral complications can be envisaged. Further research, however, is necessary to correlate their expression with the activation of specific molecular pathways in different T and B cell subsets in order to increase our knowledge and ultimately improve the efficacy of our immunosuppressive strategies.

Study design

This is a comparative study, including serum samples of lung transplant recipients prospectively collected between June 2008 and December 2013 at two timepoints: time of transplantation and 12 months post-transplant. Within this period a total of 710 lung transplant recipients consented for prospective storage of biological samples including serum, plasma or BAL for scientific purposes. Written informed consent was obtained from all recipients. Among them, 252 received alemtuzumab as induction therapy and for 30 patients, serum samples for the two timepoints defined by the study protocol were available. The remaining 222 patients could not be included in the analysis for the following reasons: 1) they did not have serum samples at both timepoints, 2) the serum samples were obtained at day of listing, 3) the collected biological samples did not include serum. The 30 control patients were chosen among the 233 patients who did not receive any induction therapy and had serum samples at both timepoints. Control patients were matched based on the following variables: gender, age, underlying diagnosis, CMV risk, type of transplantation and PGD grade (Table 1).

Table 1

– Maintenance immunosuppression protocol
Time after Tx	Tacrolimus (target trough level)		Steroids		Mycophenolate mofetil
	No induction	Alemtuzumab	No induction	Alemtuzumab	No induction	Alemtuzumab
0-3 months	15-18 ng/ml	10-12 ng/ml	0.3 mg/kg	0.2 mg/kg	1-1,5g twice a day	-
3-6 months	13-15 ng/ml	8-10 ng/ml	0.2 mg/kg	0.15 mg/kg	1-1,5g twice a day	-
6-12 months	10-12 ng/ml	6-8 ng/ml	0.15 mg/kg	0.1 mg/kg	1-1,5g twice a day	-
12-24 months	8-10 ng/ml	5-7 ng/ml	5 mg/d	5 mg/d	1-1,5g twice a day	1-1,5g twice a day

Inclusion criteria were primary lung transplantation, adult age and availability of serum samples both at time of transplantation and 1 year after transplantation. Exclusion criteria were multi-organ transplantation, retransplantations and pediatric age. No patient had signs or diagnosis of acute cellular rejection, antibody-mediated rejection, infection or CLAD at time of blood sampling. All experiments and methods were performed in accordance with relevant guidelines and regulations. The study has been approved by the Institutional Ethical Committee of the Medical University of Vienna [ECS 1729/2020] and was conducted according the declaration of Helsinki.

Clinical protocol and definitions

Lung transplant recipients received either alemtuzumab (Genzyme/Sanofi, Cambridge, USA) as a single intravenous dose of 30 mg after LTx at arrival at the intensive care unit (ICU) or no induction therapy. Recipients after alemtuzumab induction received a low-dose CNI-based maintenance immunosuppression, while recipients without induction therapy received a triple-drug protocol (IS protocols and target blood levels are shown in Table 1). During follow-up, in case of deterioration of kidney function, infections requiring hospital admission or rejection, the target blood levels were adapted to the low or high end of the respective target range (Table 1). Perioperative infectious prophylaxis was based on broad-spectrum antibiotics or adapted to resistance testing. All patients received a lifelong Pneumocystis prophylaxis with trimethoprim-sulfamethoxazole or atovaquon. Prophylactic inhalation therapy with amphotericin B and gentamycin or according to pretransplant airway colonization was provided for 1-3 months. CMV prophylaxis included CMV hyperimmunoglobulines (POD 1,7,14 and 21) together with valganciclovir for a minimum of 3 months. In high-risk patients (donor +/recipient -) a 12 months prophylaxis was performed. Surveillance bronchoscopy with transbronchial biopsy (TBB) and bronchoalveolar lavage (BAL) were performed 2 weeks and 1, 2, 3, 6, 12 months after transplantation and whenever clinically indicated. All patients received a chest computed tomography (CT) once a year. Biopsies were classified according to ISHLT criteria ⁵¹. ACR grade A2 and LB grade B2 or higher were treated with a pulse of steroids for 3 days with consecutive dose tapering. In case of inadequate clinical response, ATG (2 mg/kg) was administered for 5 days.

Quantitative real-time PCR

We analyzed the expression levels of 13 miRNAs: hsa-miR-30b-5p, hsa-miR-24-3p, hsa-miR-98-5p, hsa-miR-99a-5p, hsa-miR-23b-3p, hsa-miR-125a-5p, hsa-miR-182-5p, hsa-miR-17-5p, hsa-miR-486-5p, hsa-miR-21-5p, hsa-miR-146a-5p, hsa-miR-155-5p and hsa-miR-31-5p. These miRNAs have been selected because previously found involved in regulating the proliferation and function of main immune cell types of the innate and adaptive immunity as well as in the pathogenesis of allograft rejection. Total RNA was isolated from serum samples using miRNeasy Serum/Plasma Kit (Qiagen). RNA concentration and purity were evaluated using a spectrophotometer. Total RNA was then reverse transcribed with the miRCURY LNA RT Kit (Qiagen) according to the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) was performed with LNA probes (miRCURY LNA miRNA PCR Assay, Qiagen) and miRCURY SYBR Green PCR Kit (Qiagen) for all miRNAs. Expression levels of RNU6 were used as the normalization control. All reactions were performed on an LC480 Real-Time PCR system (Roche Diagnostics) according to the manufacturer’s recommendations. Each experiment was performed in triplicates. The threshold cycle (Ct) was defined as the fraction cycle number at which fluorescence exceeded the given threshold. Relative gene expression level quantification was compared with internal standards and analyzed using the 2^−ΔΔCt method.

Cytokine expression by flow cytometry

A total of 17 cytokines were isolated from 1:1 diluted serum samples using the LEGENDplexTM Human Th Cytokine Panel (12-plex), the LEGENDplexTM Human B Cell Panel (13-plex) and Human Immune Checkpoint Panel 1 (12-plex) as per the manufacturer's instruction. These panels are bead-based multiplex assays, which use fluorescence-encoded beads. Quantification was performed using BD LSRFortessa™ ▢ Cell Analyzer. Each patients’ sample was measured in duplicates. Results were analyzed using LEGENDplexTM Software.

In-silico data analysis

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed using R version 4.0.5. Genes targeted by miRNAs were identified using the multiMiR package. First, the “get_multimir” function was used to retrieve all targeted genes. The analysis was limited to experimentally validated miRNA-target interactions. MiRNA-target pairs with support type “negative” were not considered for further analysis. Next, the remaining target genes were mapped to KEGG pathways using the R package “org.Hs.eg.db”. Finally, pathways significantly enriched in genes targeted by miRNAs were identified using Fisher’s exact test. Across all performed tests, p-values were corrected for multiple testing using the “fdr” approach. For visualization purposes, significantly enriched pathways sharing at least 10 genes were connected, and the resulting graphs were plotted using the R package “ggraph”.

Statistical analysis

Categorical variables were reported as absolute and relative frequencies (%), continuous variables as median (interquartile range, IQR) or mean (± standard deviation). Relative frequencies were calculated based on the number of patients alive in follow-up at the respective timepoint. Chi-square tests, Fisher exact tests, Mann-Whitney U-tests, or ANOVA were used to compare variables as applicable. Correlations were calculated using Spearman’s correlation test. Data was analyzed using SPSS version 26.0 software or R 4.0.5 and graphics were designed with GraphPad Prism 6.

Conflict of interests: none

Authors contribution:

Design of the study: A.B., T.W., K.H., F.M.,P. J.

Execution of the study: A.B., S.B., S.A., H.O.B., M.L.W.

Contributed important reagents: A.B., S.B., T.W., F.M., P.J.

Collected data: A.B., S.B., S.A., H.O.B.

Analyzed data: A.B., S.B., M.L.W.

Manuscript writing:A.B., M.L.W.

Manuscript editing: A.B., S.B., S.A., H.O.B., M.L.W., S.S., T.S., W.K., T.W., K.H., F.M., P.J.

Approval of final version: A.B., S.B., S.A., H.O.B., M.L.W., S.S., T.S., W.K., T.W., K.H., F.M., P.J.

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No competing interests reported.

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You are reading this latest preprint version

Differential Expression of Circulating MiRNAs After Alemtuzumab Induction Therapy in Lung Transplantation

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Abstract

Figures

Introduction

Results

Demographics

Longitudinal miRNAs expression

Comparison of miRNA expression between groups

Cytokine levels

Correlations

MiRNA gene target prediction and pathway enrichment analysis

Discussion

Methods

Study design

Clinical protocol and definitions

Quantitative real-time PCR

Cytokine expression by flow cytometry

In-silico data analysis

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1