Signaling pathways involved in hematopoiesis and immunity in by ecj13059


									Signaling pathways involved in
hematopoiesis and immunity in

             Erika Bach
            Goals of lecture

• Hematopoiesis (blood cell development) in
  embryos and larval stages

• Immunity (responses to foreign organisms
  and pathogens) in larvae and adults
Drosophila life cycle

                • Four major stages
                   –   Embryo
                   –   Larva (3 instars)
                   –   Pupa
                   –   Adult
                • Short - 9 days long
                • Easily observed
                • Powerful genetics
   Functions of blood cells

• Phagocytose apoptotic cells during
• Tissue remodeling
• Immune responses
  – phagocytosis
  – arrest microbial growth
  – secrete anti-microbial peptides
Why do flies need blood cells?
Early embryonic blood cell development

                A. Stage 5: hemocytes arise from the
                   hemocyte anlagen in embryonic
                B. Stage 11
                    – 700 prohemocytes appear 2
                      hrs after gastrulation (st.10)
                    – express the GATA
                      transcription factor serpent
                    – migrate throughout the embryo
                    – differentiate into:
                      plasmatocytes and crystal cells
Late embryonic blood cell development
                   C. Stage 17
                   • are induced to become
                   • phagocytose dying cells
                   • participate in immune
                      Crystal cells
                   • unknown function in the
   Major types of embryonic blood cells
• Plasmatocytes
   – comprise 90% of embryonic hemocytes
   – acquire phagocytic capabilities after expression of
     Croquemort, CD36, a scavenger receptor
   – act as macrophages in tissue remodeling and phagocytose
     dead or dying cells at the end of embryogenesis
• Crystal cells
   – >10% embryonic hemocytes
   – cytoplasmic crystalline inclusions
   – unknown function
 Marker of embryonic hemocytes

• Prohemocytes express Serpent (srp)
  - srp is required for embryonic hematopoiesis and
    formation of the fat body (fly “liver”)
  - srp is highly homologous to hematopoietic GATA factors

• Plasmatocytes express glial cells missing (gcm)
    - Gcm and its mammalian homologs have all been shown to
      bind the same binding site sequence with similar affinities

• Crystal cells express Lozenge (Lz)
  - Lz contains a Runt domain
  - Lz is highly homologous to AML1 protein
    aml1 is required for definitive hematopoiesis
    aml1 locus is the most frequent target of chromosome
    rearrangement in acute myeloid leukemia.
GATA Factors: Structure and function
AML/Runx proteins and their functions
srp mutants lack prohemocytes
gcm and gcm2 are required for transition
   from plasmatocyte to macrophage

                   gcm and gcm2 are required for:
                   • proliferation of plasmatocyte precursors
                   • the expression of Croquemort
                   • the ability of plasmatocytes to convert
                            into macrophages
 Lozenge is required for crystal cell

          Permissive temp Restrictive temp


Relationships between Srp, Lz and Gcm

               • In srp mutants, lz is not expressed

               • Srp and Gcm do not co-localize in a
               group of cells presumed to be CCP

               • Expression of Lz and Gcm is
               mutually exclusive

               • Mis-expression of Gcm in CCP
               results in croquemort expression

               • Gcm-expressing CCP have altered
               morphology, consistent with change in
               cell fate
Lineage of embryonic hemocytes
Evolutionary conservation of hematopoietic
        developmental programs?

        GATA-1 KO mice have no red blood cells
Mammalian hematopoiesis
Larval hematopoiesis
       • the primary organ of larval
         hematopoiesis is the lymph gland
       • prohemocytes in the lymph gland
          – self-renew and differentiate into
            specific types of hemocytes
          – hemocytes are released into the
            circulatory system, called the
          – the circulatory system in flies is open,
            and the internal organs are
            surrounded by hemolymph
Post-embryonic hematopoiesis
                  Larval hemocytes
• plasmatocytes
   – 90% of larval hemocytes, small, non-adhesive cells
   – under immune challenge or pupariation, they become activated, increase
     in number and in phagocytosis and secretion
• lamellocytes
   – Are induced to differentiate following immune challenge
   – V. large, flat cells, separate lineage from plasmatocytes
   – encapsulate objects too large to be phagocytosed which are then
     melanized by crystal cell
• crystal cell
   – 5% of larval hemocytes
   – have crystalline-like inclusion in their cytoplasm
   – separate lineage from plasmatocytes as mutants that lack crystal cells,
     e.g. lz mutants, have plasmatocytes
Signaling pathways involved in larval
 hematopoiesis: JAK/STAT pathway

                            • JAK/STAT
                              pathway is
                              essential for
                              blood cell
                            • hop and stat92E
                              are involved in
The Drosophila JAK/STAT pathway
JAK/STAT pathway in fly hematopoiesis

• gain-of-function mutations hopTum-l and hopT42 encode
  hyperactive kinases

• at permissive and restrictive temps, they exhibit over-
  proliferation and precocious differentiation of hemocytes
   – In 2nd instar hopTum-l and hopT42 larvae, lamellocytes are 10-15% of
     the total hemocytes, compared to <1% in WT
   – By 3rd instar, lamellocytes in hopTum-l larvae are 50-75% of the total
     hemocytes, compared with <5% in WT
   – plasmatocytes frequently have abnormal intracellular features
   – lamellocytes in hopTum-l and hopT42 larvae aggregate into masses that
     often become melanized, the so-called “melanotic tumors”
Melanotic tumor

           • appears as a black mass
           • consists of at least two
             cell types, plasmatocytes
             that are encapsulated by
           • the aggregate is
             melanized, presumably
             by activation of the
             cascade in crystal cells
           • strongly correlated with
Signaling pathways involved in larval
 hematopoiesis: Toll/NFkB pathway

                   • Loss-of-function mutants (Tl,
                     tb, pll) have reduced
                   • cac l-o-f mutants and Tl g-o-f
                     mutants (Tl10B) have increased
                     hemocytes and melanotic
                     tumors (like hopTum-l)
                   • JAK/STAT and Tl pathways
                     regulate genes in blood cell
Signaling pathways in involved larval
        hematopoiesis: Notch
                     • Notch l-o-f mutants have in
                       decreased crystal cells
                     • Over-expression of Notch
                       induces differentiation of
                       crystal cells
                     • Notch function is necessary
                       for lamellocyte
                       proliferation upon
                     • Notch does not play a role
                       in the differentiation of the
Innate immunity in flies and man
         What is innate immunity?
• Innate immunity concerns the detection of
  constitutive and conserved motifs in pathogens and
  the response to it.

• In contrast, in adaptive immunity foreign organisms
  are not directly sensed and acquires 4-7 days for
  clonal expansion T and B cells. (Mammals have
  acquired immunity, flies do not.)

• Thus, the innate immune system keeps us alive while
  the adaptive immune response is gearing up and also
  participates directly in shaping the adaptive immune
An important observation: the Tl pathway
 is involved in immunity in Drosophila

• B. Lemaitre, J. Hoffmann and colleagues showed
  definitely in 1996 that mutations in the Tl gene resulted in
  susceptibility to fungal infection

• This was extremely exciting because until then the Tl
  pathway had been very extensively studied as the major
  dorsal-ventral patterning pathway in the embryo.
   The Toll pathway in mammals linked
      innate and acquired immunity!

• Until 1997, immunologists had thought that innate immunity
  primary function was to keep you alive until adaptive
  immunity irradicated the pathogen
• However, Medzhitov and Janeway showed that human Toll
  receptors were a link between innate and adaptive immunity.
• They cloned and characterized a human homologue of the
  Drosophila Toll and showed that a constitutively active human
  Toll induces:
   – the activation of NF-kB
   – the expression of NF-kB-controlled genes for the inflammatory cytokines IL-1,
     IL-6 and IL-8
   – the expression of the costimulatory molecule B7.1, which is required for the
     activation of naive T cells.
How do flies mount immune responses?

• Humoral response: the fat body produces antimicrobial
  peptides which are released into the circulatory system

• Cellular response: hemocytes expand and differentiate and
  participate in phagocytosis and encapsulation

• Induction of proteolytic cascades (leading to melanization
  and coagulation) and production of signaling molecules
Innate immunity in Drosophila
                  PAMPs and PRRs

• Pathogen-associated molecular patterns (PAMPs)
  are not virulence factors
   – Produced only by microbes
   – Invariant between class of microbes
   – Essential for microbial survival
• Pattern-recognition receptors (PRRs) (in host)
   – Sense PAMPs
   – Cannot distinguish pathogen vs. non-pathogen
   – Functions include:
      • Opsonization and phagocytosis
      • Activation of complement
      • Activation of pro-inflammatory signaling cascases
    Antimicrobial peptides (AMPs)

• In response to septic injury, AMPs are produced
  rapidly and massively in the larval fat body (and also
  by hemocytes) and are released immediately into the

• AMP genes have kB sites in their promoters, and thus
  are regulated by NFkB/Rel proteins that are activated
  in response to infection.

• What other pathways are involved in the induction of
  proteolytic cascades or AMPs?
Comparison of AMPs
    NFkB-like proteins in Drosophila

• Dorsal (Dl) is involved in dorso-ventral patterning
  in the embryo (not immune responses)

• Dorsal-type immune factor (Dif) is mainly
  involved in the induction of AMP genes in
  response to fungal and gram-positive bacterial

• Relish (Rel) regulates the induction of peptides
  active against Gram-negative bacteria
Current view of larval immunity
Responses to fungi and gram+ bacteria

                • Both require the Tl pathway
                • In mice, Tl4 acts as a direct sensor
                  of microbial compounds (eg LPS)
                • However, in flies Tl is not a direct
                  sensor. In the embryo, Spz is
                  cleaved by serine proteases from a
                  “pro” form. In immunity, a
                  proteolytic cascade activates Tl
                  ligand Spaetzle
                • In immunity, the drosomycin gene
                  is constitutively on in mutants for
                  the necrotic gene (a serine
                  protease inhibitor - serpin)
How does this upstream protease cascade
     sense the pathogen in flies?

                 • Fungi - not known
                 • Gram + bacteria -
                   peptidoglycan recognition
                   protein (PGRP)
                    – Flies have 13 PGRP genes
                      and several are upregulated
                      in response to septic
                    – PGRP-SA (semmelweis) is
                      required for activation of
                      Tl after exposure to Gram
                      + bacteria
As an aside…what about the other 8 Tl
           genes in flies?
     • Flies have 9 Toll receptor genes
     • Do they all function in immunity?
Responses to gram negative bacteria

              • Immunity involves the immune
                deficient (imd) pathway.
              • Ligand is gram-negative
                peptidoglycan Diaminopimelic
                acid (DAP)
              • Receptor may be PGRL-LC
Stress-response in Drosophila

                • Upon tissue damage, what
                  are the signaling pathways
                  that are activated to restore

                • To be discussed in detail
                  on Thursday (Agaisse et al,
                  Dev Cell, 2003)
Genetic screens for immune function
ird7 mutants do not induce Diptericin or
   activate Relish in response to Gram
             negative bacteria
UAS-GAL4 tissue specific expression


   UAS-GFP              Promoter-GAL4

PGRP rescues Dipt-lacZ induction in
 response to E. coli in ird7 mutants
                   Conclusions I
• Evolutionary conversation of embryonic (and
  larval?) developmental programs of hematopoiesis -
   – key players serpent, a GATA factor and lozenge, an aml-
     like factor
   – Future work: what controls these factors in flies? Parallel
     story in vertebrates?

• Tl and JAK-STAT pathways control hemocyte
  differentiation in larva -
   – Future work: what are the ligands and receptors? How are
     the Tl and JAK-STAT pathways inter-connected?
      Conclusions: II
Fungal and Gram+ immunity
Conclusions III: Gram - immunity
                                Selected references
•   Lebestky T, Chang T, Hartenstein V, Banerjee U. Specification of Drosophila hematopoietic lineage by conserved
    transcription factors. Science. 2000 Apr 7;288(5463):146-9
•   Hoffmann JA, Reichhart JM., Drosophila innate immunity: an evolutionary perspective.Nat Immunol. 2002 Feb;3(2):121-
    6. Review
•   Lanot R, Zachary D, Holder F, Meister M. Postembryonic hematopoiesis in Drosophila., Dev Biol. 2001 Feb
•   Tzou P, De Gregorio E, Lemaitre B. How Drosophila combats microbial infection: a model to study innate immunity and
    host-pathogen interactions.Curr Opin Microbiol. 2002 Feb;5(1):102-10.
•   Lemaitre B, Nicolas E, Michaut L, Reichhart JM, Hoffmann JA. The dorsoventral regulatory gene cassette
    spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell. 1996 Sep 20;86(6):973-83.
•   Medzhitov R.Toll-like receptors and innate immunity. Nat Rev Immunol. 2001 Nov;1(2):135-45.
•   Medzhitov R, Preston-Hurlburt P, Janeway CA Jr. A human homologue of the Drosophila Toll protein signals activation
    of adaptive immunity.Nature. 1997 Jul 24;388(6640):394-7.
•   Choe KM, Werner T, Stoven S, Hultmark D, Anderson KV. Requirement for a peptidoglycan recognition protein (PGRP)
    in Relish activation and antibacterial immune responses in Drosophila. Science. 2002 Apr 12;296(5566):359-62.
•   Wu LP, Choe KM, Lu Y, Anderson KV. Drosophila immunity: genes on the third chromosome required for the response
    to bacterial infection., Genetics. 2001 Sep;159(1):189-99.
•   Ligoxygakis P, Pelte N, Hoffmann JA, Reichhart JM. Activation of Drosophila Toll during fungal infection by a blood
    serine protease. Science. 2002 Jul 5;297(5578):114-6.
•   Gottar M, Gobert V, Michel T, Belvin M, Duyk G, Hoffmann JA, Ferrandon D, Royet J. The Drosophila immune
    response against Gram-negative bacteria is mediated by a peptidoglycan recognition protein. Nature. 2002 Apr
•   Cantor AB, Orkin SH. Transcriptional regulation of erythropoiesis: an affair involving multiple partners. Oncogene. 2002
    May 13;21(21):3368-76.
•   Westendorf JJ, Hiebert SW. Mammalian runt-domain proteins and their roles in hematopoiesis, osteogenesis, and
    leukemia. J Cell Biochem. 1999;Suppl 32-33:51-8.

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