Primary and secondary immunodeficiencies of the IL-12/IFN-γ axis

Abstract

IL-12/IFN-γ axis is a principal pathway for intramacrophagic pathogens immunity such as leishmania or mycobacteria. Alterations in this axis, being both congenic (causing the primary immunodeficiency Mendelian Susceptibility to Mycobacterial Disease, MSMD) or acquired (treatment with anti-TNF-α drugs) cause susceptibility to this type of microbes. MSMD causes susceptibility mainly to non-pathogenic mycobacteria, salmonella and candida; besides, MSMD- causing mutations have been detected in Mycobacterium tuberculosis and Leishmania patients. On the other hand, the death of an anti-TNF-α in-utero exposed infant after BCG vaccination together with the effect of anti-TNF-α drugs in tuberculosis reactivation in adults and the known tole of TNF-α in B cell maturation reveal the need for an in-depth study of in-utero exposition to anti-TNF-α drugs. With that our hypothesis is that patients with extrapulmonary Mycobacterium tuberculosis infection or visceral leishmaniasis have a primary dysfunction of the IL-12/IFN-γ axis and that exposure to anti-TNF-α antibodies during whole pregnancy in children born to mothers with inflammatory bowel disease affects the normal development of the neonatal immune system, conferring a secondary immunodeficiency, which includes a dysfunction of the IL- 12/IFN-γ axis. Both extrapulmonary tuberculosis (n=23) and visceral leishmaniasis (n=24) patients presented alterations in the IL-12/IFN-γ pathway; however, we did not detect any complete defect. Concretely, the patients with extrapulmonary tuberculosis had a diminished response to IFN-γ while visceral leishmaniasis patients had a diminished production of IFN-g. Genetic study of these patients to unravel mutations causing partial forms of susceptibility to intramacrophagic infections is then needed. Besides, we detected an IL-12Rβ1 defect in a Peruvian patient that was misdiagnosed as multi-resistant tuberculosis, being a disseminated infection by the vaccine strain BCG. After the detection of the genetic defect, the patient was transferred to the National Institute of Health in the USA, where she received the appropriate treatment and the microbiological diagnosis was corrected resulting in the resolution of the infection. This case remarks the fact that suspicion of this forms of immune deficiency and their detection changes the prognostics and outcome of the patient. The study of the effect of anti-TNF-α on the exposed infant immune system (n=7) revealed a T and B cell maturation defect that was corrected at 12 months, normal cell proliferation after mitogen stimulation and normal immunoglobulin production and vaccine response without an increase of severe infections. On the other hand, Treg cell frequency was low in exposed infants, without reaching normalization at 12 months of age. Treg cell frequency in neonates inversely correlated with anti-TNF-α through level in the mother during third trimester of pregnancy and with T cell proliferation after a mild mitogen stimulation. These data with the increased atopia/allergy in the studied infants suggest the need of a long-term follow-up for Treg cells and the advent of immune dysregulation events. Antimycobacterial response was diminished in exposed infants and not totally recovered after washing the drug from the blood in the culture. On the other hand, coinciding with the decrease of the drug levels in blood, the production of IL-12, IFN-γ and TNF-α increased. We conclude that the effects of anti-TNF-α exposure during pregnancy are not permanent and that BCG vaccination in these population should be avoided until, at least, 12 months of age. By last, the transition between the intra- and extra-uterine world is a special life-situation where the immune system plays a major role. We studied it in healthy cord blood donors, with special

attention to the IL-12/IFN-γ pathway and B cell compartment, including regulatory B cells (Breg). Breg cells, defined as CD24hiCD38hi B cells, were expanded in cord blood, with capacity to produce IL-10 and to inhibit IL-4 and IFN-γ production by T cells with a similar phenotype when compared with adult Bregs. Besides, response to mycobacterial challenge was diminished. Interestingly, the diminished production of IFN-γ was associated with Breg cell frequency, opening the door to new research studying the role of these cells in different neonatal conditions as well as in cord blood derived stem cell transplantation.

Esta tesis explora la vía de IL-12/IFN-g, central en la inmunidad a gérmenes intramacrofágicos, en el contexto de defectos primarios y secundarios. Los defectos primarios en esta vía causan susceptibilidad mendeliana a las micobacterias (MSMD), una inmunodeficiencia primaria que cursa con susceptibilidad a micobacterias no patogénicas principalmente, pero en la que se han descrito pacientes con infecciones por Mycobacterium tuberculosis y con leishmaniasis. En este escenario, la hemos estudiado en pacientes pediátricos con tuberculosis extrapulmonar y leishmaniasis visceral, revelando que no existían defectos completos de la vía, pero sí una alteración funcional en ésta en los dos grupos de pacientes estudiados. Esto reveló la necesidad de un estudio genético exhaustivo para revelar defectos parciales causantes de esta susceptibilidad. El diagnóstico la deficiencia de IL-12Rβ1 en una niña con infección diseminada por BCG, inicialmente diagnosticada como tuberculosis multirresistente, permitió el tratamiento adecuado que llevó a su curación, mostrando la relevancia del diagnóstico temprano del MSMD.

Por otro lado, el hecho que se describiera un caso de muerte tras la vacunación con BCG de un neonato expuesto a fármacos anti-TNF-α durante el embarazo hizo pensar que la exposición a estos fármacos durante el embarazo pudiera llevar a defectos en el sistema inmunitario del neonato. Tras su estudio, observamos una inmadurez transitoria del compartimiento B y T; por otro lado, la disminución de la frecuencia de células T reguladoras que no normalizó con la edad juntamente con un aumento de la presencia de atopia o alergia en este grupo. Además, observamos una disminución de la respuesta a micobacterias en los niños expuestos, que mejoró con la edad. Concluimos que los efectos de los niños expuestos a anti-TNF-α durante el embarazo no parecen ser permanentes y que la vacunación con BCG de esta población debe ser evitada hasta los 12 meses de edad.

El estudio de sangre de cordón de neonato sano reveló un aumento de la población de células B reguladoras. Además, la frecuencia de estas células se asoció inversamente con la producción de IFN-γ tras el estímulo con micobacterias, que se encontró disminuido en el neonato. Abriendo la puerta a nuevas investigaciones para estudiar su papel en diferentes condiciones del neonato, así como en el trasplante de progenitores hematopoyéticos.